Use of Nigella sativa to increase immune function

ABSTRACT

A pharmaceutical composition containing an extract of the plant Nigella sativa is disclosed for treating cancer, preventing the side effects of anticancer chemotherapy, and for increasing the immune function in humans.

FIELD OF THE INVENTION

The present invention is generally directed to the fields of medicineand pharmacology, and specifically directed to using the plant seedextract of Nigella sativa Linn (N. sativa) in the treatment of cancer,viral diseases, protection from side effects of chemotherapy and growthfactor for bone marrow in hematopoiesis.

CITED REFERENCES

A full bibliographic citation of the references cited in thisapplication can be found in the section preceding the claims.

DESCRIPTION OF THE PRIOR ART

A variety of herbal and plant extracts or preparations are availabletoday for treating any number of diseases affecting the human body. Somepreparations have been known for literally thousands of years whileothers are just being discovered to have curative effects. Effectiveplant extracts are highly desired as a "natural" way to treat a disease.It is believed that natural preparations will not have as much of anadverse effect on the body as synthetic preparations.

One of the primary targets for treatment is cancer. Anticancer remediesare available today which are effective in killing cancer cells.However, many of these medicaments also damage or kill off normal cellsor have other serious side effects. It is therefore vitally important todevelop an anticancer program which is specific for the cancerous growthin a body, but which is not toxic to the rest of the body system.Ideally, the program will include treatment using natural plantextracts. As used in this disclosure, the term "body" or "patient" caninclude any warm-blooded mammal, but is specifically intended to referto the human body.

The medicinal properties of various spices and herbs in general is known(Srivastava, 1989). U.S. Pat. No. 4,986,895 to Grossman et al. isdirected to use of water-soluble plant extracts in the treatment ofvirus skin infections. U.S. Pat. No. 5,178,865 to Ho et al. is directedto the use of Chinese herbal extracts in the treatment of HIV relateddisease in vitro. A total of 56 herbal extracts were screened foranti-HIV activity using in vitro techniques.

Aruna (1990) also describes the use of spices, leafy vegetables andcondiments having diverse medicinal properties. Products of 20 spices orleafy vegetables were screened for anti-carcinogenic activity usinginduction of glutathione-S-transferase. One of the plants utilized wasCuminum cyminum Linn (C. cyminum), also known as cumin. All of thespices and leafy vegetables were tolerated well and no toxic effectswere seen.

Bitterman et al. (1991) disclose a study that was performed on apopulation of 964 adult patients, of which 28% suffered from malignantdiseases of the urinary tract and 72% from a wide spectrum of the nineneurologic diseases. The results conclude that the use of C. cyminum inthe diet may contribute to the prevention of diseases mediated byperoxidation of lipids.

Aruna and Sivaramakrishnan (1992) reported on anticarcinogenicproperties of some spices. Cumin seeds (C. cyminum Linn) were studied.Cumin seed significantly inhibited some carcinogenesis.

Another plant extract from the plant N. sativa has shown a wide range ofmedical use. N. sativa is an annual herb belonging to familyRanunculaceae. Other species of Nigella include Nigela arvensis andNigella damascena. Induction of callus cultures indicates considerablechromosomal variations in callus tissues between the different speciesof Nigella (Datta, et al. 1983).

N. sativa is characterized by an erect branched stem and alternatefinely divided, feathery, grayish-green leaves. The bluish-white,star-shaped flowers are terminal and solitary. Petals are absent. Thefruit is a globose capsule with small, black, rough seeds. The plant iscultivated in India, Bangladesh, Turkey, Middle-east and theMediterranean basin mainly for its seeds or "black cumin" which isalmost entirely used for edible and medical purposes, such as spices andfor treatment of various diseases.

The ripe seeds of N. sativa, also known as Kalajira or Kalaonji, areknown to have a wide range of medicinal uses (Kirtikar et al. 1982, andChopra et al. 1982). The constituents of the seeds include saponin, anessential oil, a bitter compound (nigellone) and tanners. Thesesubstances have been shown to have diuretic (Nadkarni 1976), cholagogicand antispasmodic (Tennekoon, et al. 1991), carminative (Shayeb andMabrouk, 1984), galactogogic (Vihan 1987), antibacterial (Hassan, et al.1989), antifungal (Agarwal, et al 1979), anthelminthic (Akhtar 1991) andemmenagogic (Siddiqui et al. 1988) properties. al-Awadi, et al. (1985)have demonstrated an antidiabetic effect of N. sativa plant extract.

N. sativa has been reported to be used in Egyptian folk medicine as adiuretic and carminative (Sayed 1980). The oil is used in the treatmentof asthma, respiratory oppression and coughs. The active principle,nigellone, has been isolated from the volatile oil fraction and isreported to be useful in the treatment of bronchial asthma.

The petroleum ether extract of the seeds at 1000-62.5 parts per million(ppm) concentrations was found to have the same activities as growthregulating juvenile hormone when tested against the fifth instar larvaeof Dysdercus similis (Kumar et al. 1987).

al-Awadi et al. (1981) is directed to the study of the effect of aplant's mixture extract containing N. sativa on liver gluconeogenesis.The researchers report that non-insulin dependent diabetes mellitus istreated in Kuwait by a plant mixture extract, which contains N. sativa.In this study, a powdered mixture of equal portions of N. sativa, Linn,Commiphora myrrh, Eng, Ferula Asafoetida, Linn, Aloe vera, Linn, andolibanum was boiled in distilled water for 10 minutes. Diabetic animalswere given a daily dose by gastric intubation. The results indicate thatthe anti-diabetic action of the plant's extract is at least partlymediated through decreased liver gluconeogenesis.

Elkadi and Kandil (1987) are directed to N. sativa and its effect onhuman T-cells. N. sativa was tested in volunteers with a low helperT-cell to suppressor T-cell ratio. The results indicated an increase inthe helper T-cell population in the experimental group. Further, thehelper T-cell to suppressor T-cell ratio increased while the ratiowithin the control groups remain the same.

Nair et al. (1991) investigated the effects of N. sativa as potentialprotective agents against cisplatin-induced toxicity in mice. Someprotective effects were shown by the use of N. sativa extracts.

Salomi, N. J., et al. (1992) studied N. sativa seeds containing certainfatty acids for antitumor activities against Ehrlich ascites carcinoma(EAC), Dalton's lymphonia ascites (DLA) and Sarcoma-180 (S-180) cells.The paper presents the results of in vitro and in-vivo antitumorexperiments. The active antitumor principle was isolated. It was foundthat the active principle was cytotoxic for EAC cells, KB cells andlymphocytes.

Salomi et al. (1991) reported the effect of the active principleisolated from N. sativa in inhibiting chemically induced skincarcinogenesis. Intraperitoneal administration of N. sativa extract wasshown to prevent the incidents of soft tissues sarcomas and reducedtumor diameters in treated groups.

Metcalf (1984, 1985) reported the inhibitory effects of N. sativa onchemical carcinogenesis in mice. However, there was no evidence in thesepapers of the destruction of human tumor cells.

SUMMARY OF THE INVENTION

The present invention provides an anticancer remedy and treatment whichhas, as its active ingredient, the extract of the plant N. sativa. Whenused properly, the medicament of the present invention is useful intreating cancer, preventing toxicity of anticancer drugs in human bodyand in increasing immune function.

The present invention is specifically directed to a pharmaceuticalcomposition for the treatment of cancer comprising a pharmaceuticalpreparation consisting essentially of an extract from Nigella sativa, ata concentration which is effective to destroy cancer cells in a patient.

Additionally, the present invention is directed to a pharmaceuticalcomposition for the treatment of the side effects of anticancer therapyconsisting essentially of an extract from Nigella sativa, at aconcentration of the extract which is effective to reduce the sideeffects of anticancer therapy.

The present invention is also directed to a method for treating humanssuffering from the side effects of anticancer chemotherapy using theextract of Nigella sativa, comprising administering to humans effectivedoses of the composition described above.

Further, the present invention is directed to a method for increasingthe immune function in humans comprising administering to humanseffective doses of an extract from Nigella sativa, at a concentration ofthe extract which is effective to increase the immune function.

The present invention is also directed to a method for protecting thenormal cells from cytopathic effects of virus, comprising administeringto humans effective doses of an extract from Nigella sativa, at aconcentration of the extract which is effective to protect against thecytopathic effects of the virus.

The present invention is still further directed to a method forincreasing antibody producing B cells, comprising administering tohumans an extract from Nigella sativa, at a concentration which iseffective to increase the antibody-producing B cells.

The present invention is also directed to a process for inhibiting tumorcells without affecting normal or nontumor cells in a patient comprisingadministering to a patient an extract from Nigella sativa, at aconcentration which is effective to inhibit the tumor cells withoutaffecting nontumor cells.

The present invention is also directed to a method for stimulating bonemarrow formation in humans comprising administering to humans effectivedoses of an extract from Nigella sativa, at a concentration of theextract which is effective to stimulate bone marrow formation.

In general, N. sativa extract helps stimulate bone marrow cells,protects the normal cells from cytopathic effects of virus, destroystumor cells and increases antibody producing B cells. It protects thebone marrow against chemotherapy and at the same time, can act as ananticancer agent. All these factors makes N. sativa seed extract anideal candidate to be used as vaccine for cancer prevention and cure.

N. sativa plant extract is more effective than standard chemotherapeuticanti-cancer drugs. N. sativa extract stimulates bone marrow cells,protects the normal cells from cytopathic effects of virus, destroystumor cells and increases antibody producing B cells. Further, itprotects the bone marrow against chemotherapy and can act as ananti-cancer agent.

N. sativa plant extract also has been found to help restore immunecompetent cells in immunosuppressed cancer patients and to overstimulatebone marrow formation in normal individuals.

N. sativa extract helps free tumor antigen binding sites on B cells. Theadministration of the N. sativa extract, rather than the increase of theimmune competent cell number, has been found to help free tumor antigenbinding sites on B cells, thereby elevating the CD19 and associated cellpopulation. When the antigen binding site on the immunoglobulin moleculeon the surface of B cells is free because of N. sativa treatment, itbinds to the tumor associated antigen thereby generating an immuneresponse against the antigen.

Protection of Human Amniotic "WISH" cells from cytopathic effects ofvesicular stomatitis virus (VSV) was also observed upon administrationof N. sativa plant extract. Additionally, the serum interferon level isfound to increase; and, hence, the plant extract of N. sativa hasinterferon-like antiviral activity. This is an example of interferonlevel increasing in the circulation, preventing viral diseases and, inaddition, possibly curing viral diseases.

N. sativa promotes anti-tumor activity. Data from pharmacosensitivityscreening indicates anti-tumor activity of N. sativa plant extractmainly against melanoma and colon cancer types. N. sativa plant extractdestroys tumor cells and leaves normal cells alone, possibly because ofits ability to bind to cell surface asialofeutin (lectin) in diseasedcells, which causes aggregation and clumping of tumor cells. It alsoblocks enzymes and inappropriate gene products involved in nucleic acidsynthesis and metabolism.

Further objects, features and advantages of the invention will beapparent from the following detailed description when taken inconjunction with the accompanying drawings.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph illustrating the elevation in CD19, HLADR, NKCD3-/CD56+ and CD38 populations of peripheral blood cells from cancerpatients upon incubation with N. sativa extract over a 18 hour period at37° C. in 5% CO₂ incubator as shown in Experiment 2.

FIG. 2 is a graph illustrating the percent protection of 3.5×10⁴ WISHcells from the cytopathic effects (CPE) of vesicular stomatitis virus(VSV) by serial dilutions of N. sativa extract as described inExperiment 3.

DETAILED DESCRIPTION OF THE INVENTION

Definitions:

The following definitions will be used for the present application:

B cells: B cells or B lymphocytes secrete proteins/antibodies thatprotect the human body against infections.

CD3: Cluster Differentiation 3. These are the antibodies which indicateActivated T lymphocytes which are used by the body for its protectionagainst foreign harmful germs.

CD19: Cluster Differentiation 19. These are antibodies which help detectB lymphocytes. Elevation in CD19 indicates an elevation in B lymphocytesand vice versa.

CD56: Cluster Differentiation 56 are antibodies which inhibit NaturalKiller target cell interactions in certain systems.

G-CSF: Granulocyte-Colony Stimulating Factor

GM-CSF: Granulocyte Macrophage-Colony Stimulating Factor.

HLADR: Human Leukocyte antigen DR. DR designates a genetic locus or theantigen of the major histocompatibility complex corresponding to thelocus. An increase in HLADR indicates an elevation in immunologicalparameters against the disease.

NK: Natural Killer cells. NK is an indication to detect the NaturalKiller cells.

NKCD3-/CD56+: Natural Killer Cluster Differentiation 3-/ClusterDifferentiation 56+.

Preparation of N. sativa Extract:

The process for preparing the N. sativa extract comprises grinding theseeds and separating the extract of the N. sativa with appropriatesolvents such as alcohol or water, removing lipids by extraction withether or petroleum ether, crystallization or chromatographicfractionation and then mixing its components in the desired proportion.The N. sativa extract can then be prepared in 3 forms--oil, fluid andcrystal--by processes known to the art. The preferred process for thepreparation of N. sativa extract is described in Experiment 1.

Administration:

The extract of N. sativa may be administered by itself or in admixturewith an appropriate excipient or carrier. The preparation may beadministered to the patient by enteral, such as oral or rectal, andparenteral, such as intraperitoneal, intramuscular, intravenous orsubcutaneous route. The preparation may also be administered incombination with supplements, such as antiviral agents, immunemodulators, antibodies, other chemotherapeutic agents, or combinationsthereof. The preparation may additionally be administered in dosageform, such as by capsules, tablets, suppositories or the like.

Dosages:

N. sativa has been found to be most effective when administered at adosage of 30 g per day, with an effective range of 20-40 g per day.

N. sativa extract is also known to confer protection of human amniotic"WISH" cells against cytopathic effects of vesicular stomatitis virus(VSV). The plant extract at a dilution of 1:1000 when incubated with3.5×10⁴ WISH cells for 18 to 24 hours at 37° centigrade gave maximumprotection.

Theoretically, a patient weighing 70 kg has about 7×10¹³ cells in itsbody and N. sativa will be useful at a dosage between about 20 and 40 gper day and preferably about 30 g per day to protect against viralattack in virus endemic areas.

The invention is further illustrated by the following experiments andtests but not limited by the following experiments:

EXPERIMENTS

To evaluate the usefulness of N. sativa for cancer treatment, theactivity of the N. sativa extract was measured on bone marrow andperipheral white blood cells. In vitro antiviral antitumor and growthfactor like activity were also measured.

N. sativa extract was incubated with bone marrow cells to determine thegrowth of the cells. The results were compared with that of bone marrowgrowth factors and biological response modifiers (GM-CSF, G-CSF,erythropoietin, interferon, IL-2, and STS). Mouse connective cells andhuman amnion or "WISH" cells were also assayed.

The following calculations were used in the examples:

Calculation of Percent Elevation: ##EQU1## in which R_(T) is the averagecell count of the experimental well with extract/growth factors, R_(C)is the average background cell count with control, R_(MAX) is theaverage maximum elevation without extract/growth factor.

Calculation of Percent Inhibition: ##EQU2## in which R_(T) is theaverage cell count of the experimental well with extract/Abrin control,R_(C) is the average background cell count with control, R_(MAX) is theaverage maximum inhibition without extract/Abrin control.

Following are descriptions of some procedures used in the experiments.Universal safety precautions, known to the art, must be observed whenhandling all biological materials.

Procedure 1 Flow Cytometry Direct Immunofluorescence Staining

Automated Flow Cytometry (FC) provides an efficient, sensitive andquantitative method to analyze cell populations, sub-populations, andtheir components in suspension. Cells express and shed surface antigensthroughout their life indicating their classification, stage ofmaturation, activation state and disease state. Monoclonal antibodies(mABs) have been developed that specifically bind these surfaceantigens. Established clinical applications are in leukemia and lymphomadiagnosis, T-cell subset analysis, monitoring transplant rejection,monitoring the effects of chemotherapeutic agents on different celltypes and measuring cellular activation.

Whole blood, bone marrow or cellular suspension is first treated with ared blood cell lysing solution, washed, and then mixed with a labeledmAB against a specific membrane antigen. The direct staining procedureis then followed by analysis with the FACSCAN Immunocytometry Systemsflow cytometer (FACSCAN Users' Guide, Becton Dickinson).

All specimens are generally labeled with the following information: 1)Patient name; 2) Date drawn; 3) Time drawn; 4) Phlebotomist's initials;and 5) Panel number. Patient specimens are identified by the nurse orphlebotomist generally by checking the wristband to verify the name ofthe patient. A unique tracking number is assigned to each individualspecimen to assure proper specimen tracking.

Peripheral Blood is aseptically collected in an appropriate containersuch as a lavender top (EDTA anti-coagulant) vacutainer tube anddelivered at room temperature preferably within 48 hours. The minimumamount required is approximately 2 ml whole blood per panel ordered. Theoptimum amount required is approximately 3 ml whole blood per panelordered

The bone marrow (BM) specimen is aseptically collected in a containersuch as a lavender top (EDTA) vacutainer tube and transported at roomtemperature within 48 hours. The minimum amount required isapproximately 2 ml of BM specimen per panel ordered. The optimum amountis approximately 3 ml of BM specimen per panel ordered.

Solid tumors include those from the breast, lymph node, colon, ovarian,lung, and skin. Reference is made to the HTCA PharmacosensitivityProcedure (Procedure 5, infra.) for a description of the procedures. Thetumor should be delivered at room temperature within 48 hours ofextraction. The minimum amount required is approximately 2 ml of cellsat a concentration of 1×10⁶ /ml per panel ordered. The optimum amount isapproximately 3 ml of cells at a concentration of 1×10⁶ /ml per panelordered.

Materials:

The materials required are as follows:

EDTA Vacutainer Blood Collection Tubes

12×75 mm plastic test tubes

Serofuge

FACSCAN Flow Cytometer

Micropipettes (20 ul and 100 ul)

Micropipet Tips

Repeater Pipet

Small Beakers

Gauze Squares

Monoclonal Antibodies

Lysing Solution

PBS

0.5% p-Formaldehyde

Transport Media

50 ml Conical Tubes

Repeater Pipet Tips

Safety Shield

Sorvall TR6000 centrifuge

Coulter Cytotrol control cells

Reagents:

The reagents are prepared as follows:

A. Monoclonal antibodies--see package inserts for proper reconstitutionand storage of various mABs. For Coulter mABs, reconstitute according topackage insert and then make a 1:2 dilution in sterile water and placein an appropriate container.

B. Lysing solution--(Becton-Dickinson order #92-0002). To prepare 1×working solution, add 10 ml 10× lysing stock solution to 90 ml distilledwater. Mix well. Store at room temperature. Discard after one week.

C. Phosphate Buffered Saline (PBS) without Ca++ and Mg++ (Gibco order#310-4190AJ)

D. 0.5% Paraformaldehyde. Dissolve 2.5 g paraformaldehyde (BakerS-898-7) in 500 ml of 1× PBS without Ca++ or Mg++.

E. Coulter Cytotrol control cells. Reconstitute Cytotrol control cellsaccording to manufacturers' package insert. These cells must be preparedfresh daily.

Quality Control:

A leukogate and a negative control tube must be run for each patient.Tubes for Cytotrol control cells should be included for each mAB usedthat day.

Procedure:

Label test tubes with patient name and appropriate mAB. Using amicropipet, to each tube add 20 ul of the appropriate antibody. Using amicropipet, to each tube add 100 ul of the patient whole blood. Vortexeach tube. Incubate the tubes at room temperature for 15 minutes. Usingthe repeater pipet (set on 4) add 2 ml of 1× lysing solution to eachtube. Incubate the tubes at room temperature for 10 minutes. Centrifugetubes in serofuge set on high for 3 minutes or in Sorvall TR6000 for 5minutes at 3000 RPM. Pour off supernatant in waste beaker, being carefulnot to splash. Blot top of tube on gauze square. Vortex each tube tobreak pellet (use safety shield). Using repeater pipet (set on 4) add 1ml of 1× PBS to each tube and vortex each tube (use safety shield).Centrifuge tubes in a serofuge set on high for 3 minutes .or in SorvallTR6000 for 5 minutes at 3000 RPM. Pour off supernatant in waste beakerbeing careful not to splash. Blot top of tube on gauze square. Vortextubes to break pellet (use safety shield). Using repeater pipet (set on2) add 0.5 ml of 0.5% paraformaldehyde. Vortex. Keep tubes covered withparafilm and in the refrigerator until ready for flow cytometricanalysis.

Specimen Analysis:

Analyze each tube on the FACSCAN flow cytometer using Simulset softwarefor surface marker evaluation. (Refer to FACSCAN Users' Guide andSimulset Software Manual.) Use the complete blood count (CBC)information (be sure CBC was drawn same date and time as FC sample) toenter the total white blood count and % lymphocytes.

Procedure 2 Colony Forming Cells

In order to be able to calculate the potency of the bone marrow, thestem cell assay is performed. Information on the status of ColonyForming Units (CFU) before therapy is performed is obtained. This willallow one to precisely define the medication according to the possibletoxicities developed. Also, this will allow the prediction of thetoxicity of the medication. The character of CFU when stem cells aretreated with different medications will help determine predominance ofdevelopment of cell lines. It has been demonstrated that interferonprolongs myelopoietic differentiation; therefore, the number of CFU willincrease. This will demonstrate efficiency of interferon on bone marrowrecovery and confirm the protective activity of interferon to the bonemarrow.

With different hemopoietic growth factors, CFU development for thedifferent cell lines can be directed.

Specimen Requirements, Collection and Handling:

Specimen Requirements: Bone marrow sufficient to yield a minimum of 4.0ml and an optimum of 5.0 ml mononuclear cells at a concentration of1.0×10⁶ cells/ml.

Bone Marrow collection procedure: Prepare 4-6 50 ml Falcon tubescontaining 20 ml of tissue transport media. Transport the media in thecooler with frozen ice chips to the procedure. A mask, gloves and gownmust be worn during the procedure. Inject 2500 units ofpreservative-free heparin into the media tubes using sterile technique.This must be done no earlier then 15 minutes before obtaining the bonemarrow specimen. Mix well. When bone marrow is handed to thetechnologist in a syringe, carefully inject into the media tubes,rinsing with media twice. Discard syringe in the Sharps container. Captube and mix well. Label the specimen with the following: 1) Patient'sfull name; 2) Date specimen was received; 3) Time specimen was received;and 4) Initials of technologist who obtained the specimen. Place incooler with ice chips for transportation.

Materials:

The following materials are necessary:

Ficoll-Hypaque

Capped Test Tubes [Sterile] 15 ml

Falcon Tubes-50 ml

Sterile Laminar Flow Hood with UV Light

Centrifuge

Sterile Disposable Pipettes

1.0 ml

5.0 ml

10.0 ml

25.0 ml

RPMI 1640 [1×]

Iscove's Modified Dulbecco's Medium [1×]

Fetal Bovine Serum

Penicillin/Streptomycin

L-Glutamine [100×]

Trace Elements [100×]

2-Mercaptoethanol

Insulin Transferrin-Sodium Selenite Media Supplement (ITS)

Glass Test Tubes 12×75 ml

Trypan Blue Stain (0.4%)

Hemocytometer With Cover Slips

Microscopes (Light, Inverted)

Tissue Culture Flasks-25 CM²

Somatostatin-50 μg/ml (Sandoz-Sandostatin)

Interferon-3×10⁶ U/ml (Roferon A-Hoffman LaRoche)

Interleukin-2 (Boehringer Mannheim GMBH Cat #799068)

GM-CSF (Amgen Cat #13050)

G-CSF (Amgen Cat #10050)

Epogen (Amgen Cat #06050)

PIXY 321 (Immunex)

CO₂ Incubator

Hand Counter

Ice Chips

Media and Reagent Preparation:

After preparation of each media, the final product is labeled with thefollowing: 1) Name of Product; 2) Preparation Date; 3) Expiration Date;4) Storage Recommendations; and 5) Technologist's Initials. 10% RPMI1640 (1×) G,G Fortified:

    ______________________________________                                        AMOUNT           COMPONENT                                                    ______________________________________                                        500.0 ml         RPMI 1640 (1X) with L-Glutamine                              50.0 ml          Fetal Bovine Serum (Qualified, Heat                                           Inactivated)                                                 5.0 ml           L-Glutamine (100X), 200 mM                                   1.5 ml           Penicillin/Streptomycin                                      2.0 ml           Trace Element Mix (100X),                                                     Lyophilized                                                  0.5 ml           2-Mercaptoethanol                                            5.0 ml           ITS Media Supplement, Lophilized,                                             Gamma-Irradiated                                             ______________________________________                                    

All components are combined under a sterile laminar flow hood, andsterilized by filtration using a 0.22 micron membrane filter with a 60micron prefilter. The prepared media is labeled and stored at 2°-10° C.

IMDM/RMPI 1640 [1×] (Tissue Transport Media):

    ______________________________________                                        AMOUNT              COMPONENT                                                 ______________________________________                                        500.0 ml            RPMI 1640 [1X]                                            500.0 ml            IMDM [1X]                                                 100.0 ml            FBS                                                       5.0 ml              Penicillin/Streptomycin                                   10.0 ml             L-Glutamine (100X)                                        5.0 ml              Trace Element Mix (100X)                                  5.0 ml              Nonessential Amino Acids                                                      (100X)                                                    0.5 ml              MEM Vitamins                                              0.5 ml              2-Mercaptoethanol                                         5.0 ml              ITS                                                       ______________________________________                                    

All components are combined under a sterile laminar flow hood, andsterilized by filtration using a 0.22 micron membrane filter with a 60micron prefilter. The prepared media is labeled and stored at 2°-10° C.

Interferon:

Use 1 vial of Roferon A Interferon available from Hoffmann-LaRoche Inc.(3,000,000 U). Take 1 ml Interferon and add 39 ml of RPMI(1×) media toyield a concentration of 75,000 U/ml. Make 40 1 ml aliquots of thisdilution and freeze. For the Colony Forming Assay use 1 ml of the 75,000U/ml Interferon for each 25 cm² flask containing 4.0 ml of media and 0.5ml of cells.

Interleukin-2:

Thaw interleukin-2 and take 5 ml of interleukin-2 containing 200 unitsper ml. Immediately freeze remaining interleukin-2 into 5 ml aliquots.Add 5 ml of RPMI(1×) media to 5 ml of interleukin-2 containing 200 unitsper ml to obtain a concentration of 100 units/ml. To 10 tubes containing9 ml of the media, add 1 ml of interleukin-2 at a concentration of 100units/ml to obtain a concentration of 10 units/ml. These aliquots arestable for 30 days at 4°-8° C. For colony forming assay, use 0.5 ml (or5 units) for each 25 cm² flask containing 4.5 ml of medium and 0.5 ml ofcells.

Somatostatin:

Somatostatin is available in single vials containing 50 μg/ml and 100μg/ml from Sandoz Ltd. Store at 4°-8° C. For the Colony Forming Assay,use 1.0 ml of STS containing 50 μ/ml for each 25 cm₂ flask containing4.0 ml of media and 0.5 ml of cells. This procedure is written for 50μg/ml vials. If 100 μg/ml vials are used, use 0.5 ml of Somatostatin and4.5 ml of media for a 25 cm² flask.

GM-CSF:

Dilute 250 ul of GM-CSF containing 50,000 units in 50 ml of media toobtain 1000 units/ml. Prepare aliquots of 5 ml each containing 5,000units in sterile tubes with caps. Label each aliquot: GM-CSF--5,000units (1,000 units/ml). Close nine of these aliquots with sterileadhesive and store at 4° C. Aliquot #10, containing 1,000 units/ml(total 5,000 units), will be used as follows: Pipet one ml into each of5 sterile tubes. Add 19 ml of media to obtain 50 units/ml. (Each tubewill contain a total of 1,000 units). Label each of these aliquots:GM-CSF--50 units/ml (total 1,000 units). Close 4 of these aliquots withsterile adhesive and store at 4° C. Aliquot #5 will be marked "IN USE"and stored at 4° C. For colony forming assay, use 50 ul (or 2.5 units)for 25 cm² flask containing 5.0 ml of medium and 0.5 ml of cells.

G-CSF:

Dilute 125 ul of G-CSF, containing 60,000 units in 50 ml of media toobtain 1200 units/ml. Prepare aliquots of 5 ml each containing 6,000units in sterile tubes with caps. Label each aliquot: G-CSF--6,000 units(1,200 units/ml). Add the date. Close 9 of these aliquots with sterileadhesive and store at 4° C. Aliquot #10 containing 5 ml of 1,200units/ml (total 6,000 units), will be used as follows: Pipet one ml intoeach of 5 sterile tubes. Add 19 ml of media to obtain 60 units/ml (eachtube will contain a total of 1,200 units). Label each of these aliquots:G-CSF--60 units/ml (total 1,200 units). Add the date. Close 4 of thesealiquots with sterile adhesive and store at 4° C. Aliquot #5 will bemarked "IN USE" and stored at 4° C. For colony forming assay, use 50 μl(or 3 units) for each 25 cm² flask containing 5.0 ml of media and 0.5 mlof cells.

Epogen:

To one ml of epogen containing 2,000 units, add 19 ml of media to yield20 ml of 2,000 units or 100 units/ml. Aliquot 2 ml into each of 10cryovials. Label as follows: Epogen--100 units/ml and date. Store at4°-8° C. For colony forming assay, use 50 μl (or 5 units) for each 25cm² flask containing 5.0 ml of medium and 0.5 ml of cells.

PIXY 321-GM-CSF/IL-3:

Using 15 ml sterile capped tubes, dilute 1 ml of Pixy 321 (be sure toquantitatively transfer by rinsing vials) with 9 ml of 10% BSA and mixwell. This will yield a neutralizing effect of 1.0×10⁶ units/ml. Aliquot1.0 ml of this solution of 1×10⁶ units/ml into each of 9 cryovials.Label each vial with: name, concentration and expiration date. Store at-70° C. To the remaining 1 ml of 1.0×10⁶ units/ml, add 9 ml of 10% BSAand mix well. This will yield a neutralizing effect of 1.0×10⁵ units/ml.Aliquot 1.0 ml of this solution of 1×10⁵ units/ml into each of 9cryovials. Label each vial with: name, concentration and expirationdate. Store at -70° C. To the remaining 1 ml of 1.0×10⁵ units/ml, add 9ml of 10% BSA and mix well. This will yield a neutralizing effect of1.0×10⁴ units/ml. Aliquot 1.0 ml of this solution of 1×10⁴ units/ml intoeach of 9 cryovials. Label each with: name, concentration, andexpiration date. Store at 70° C. To the remaining 1 ml of 1.0×10⁴units/ml, add 9 ml of 10% BSA and mfx well. This will yield aneutralizing effect of 1.0×10³ units/ml. Aliquot 1.0 ml of this solutioninto each of 10 cryovials. Label each with: name, "Working Pixy,"concentration, and expiration date. Store at -70° C. One vial may bethawed and stored at 4° C. for use in the assay. Take 1 ml aliquot of1.0×10³ units/ml out of the -70° C. freezer and thaw. This will bemarked "in use" and stored at 4° C. For Colony Forming Assay, use 50 μL(or 2.5 units) for 25 cm² flask.

PROCEDURE

Sample Processing:

Using a sterile pipet, dispense 20-25 ml of histopaque into theappropriate number of capped sterile 50 ml conical tubes. Using anothersterile pipet, gently layer the bone marrow at a 45° C. angle onto thehistopaque using a 1:1 ratio of histopaque to specimen. Centrifuge at1600 RPM for 20 minutes in refrigerated centrifuge at 4° C. (brake on2). Remove and discard the supernatant. Remove the cell interface andplace in RPMI(1×) in a pre-labeled tube. Wash cells twice with RPMI(1×)media and centrifuge at 1600 RPM for 5 minutes with a brake of 2. Removeand discard the supernatant. Resuspend the cell pellet in 5 ml ofRPMI(1×) medium.

Perform cell count:

In a 12×75 ml glass tube, combine:

0.1 ml of well-mixed cell suspension

0.2 ml of 0.4% Trypan Blue stain

0.7 ml of media

This is a 1:10 dilution.

Mix well and charge one chamber of the hemocytometer with the cellsuspension. Under the light microscope, count the viable cells (thosewhich have not absorbed the Trypan Blue) in the four corner 1 mm²squares. Count the total number of cells (stained and unstained) in thefour corner 1 mm² squares.

Calculation for Cell Concentration:

1. Perform the viable cell count using the following equation:

    Average number of viable cells per square×dilution factor×hemacytometer factor =cells/ml

Sample calculation: 20 Cells (average # of viable cells per square)×10⁴×10 (dilution factor)=2×10⁶ cells/ml.

2. Determine the % viability using the following formula:

    Viable cell count×100=% viability total cell count

3. Adjust volume to amount needed for the appropriate cell concentrationfor the assay (this assay requires 1×10⁶ cells/ml) using the followingformula:

    (V.sub.1 C.sub.1 =V.sub.2 C.sub.2)

For example:

V₁ =10 ml V₂ =?

C₁ =2×10⁶ cells/mlC₂ =1×10⁶ cells/ml ##EQU3## 10 ml×2×10⁶ cells/ml=V₂1×10⁶ cells/ml

20 ml=V₂

20 ml-10 ml=10 ml

Ten ml of media must be added to have a volume of 1×10⁶ cells/ml.

4. Prepare the flasks: label eight 25 cm² liquid tissue culture flaskswith the following information: 1) Patient name; 2) Tracking number; 3)Date; 4) Growth Factor or BRM.

5. To each of the 25 cm² flasks, add bone marrow cells (BMC), growthfactor or biological response modifier (BRM), and media according toTable 1:

                  TABLE 1                                                         ______________________________________                                                       Growth Factor                                                                BMC     BRM     Media                                           ______________________________________                                        BMC + M         0.5 ml    --        5.0 ml                                    (Control)                                                                     BMC + IFN + M   0.5 ml    1.0   ml  4.0 ml                                    BMC + IL2 + M   0.5 ml    0.5   ml  4.5 ml                                    BMC + STS + M   0.5 ml    1.0   ml  4.0 ml                                    BMC + GM-CSF + M                                                                              0.5 ml    50    μl                                                                             5.0 ml                                    BMC + G-CSF + M 0.5 ml    50    μl                                                                             5.0 ml                                    BMC + EPO + M   0.5 ml    50    μl                                                                             5.0 ml                                    BMC + PIXY + M  0.5 ml    50    μl                                                                             5.0 ml                                    ______________________________________                                         NOTE:                                                                         The final volume in all flasks will be 5.5 ml.                           

NOTE: The final volume in all flasks will be 5.5 ml.

6. Incubate the flasks in the 37° C. and 5% CO₂ incubator. The flasksare to be read and fed as needed, taking care to treat each flask in theassay the same.

Reading and Reporting Results:

At days 7, 14 and 21, scan each of the flasks of the colony formingassay for colonies under the inverted microscope. A colony is defined as40 or more cells adhering to the bottom of the flask. Floating coloniesare not to be counted. Scan the entire flask and report the total numberof colonies counted for each flask on the colony forming assay reportform.

If no growth is seen at the end of 21 days, report as "No Growth" andhold the flasks for an additional 7 days. Flasks may be discarded afterthe final report is reviewed by the Medical Director.

Procedure 3 Immunomodulatory Testing

The prediction of clinical or in vitro response to cancer therapy andthe corresponding determination of optimum patient treatment through theuse of in vitro assays has been the goal of many investigators. Theimmunomodulatory assay is a procedure to determine how the patient willreact to certain biological response modifiers in vitro. It is anecessary addition to other forms of testing in that it can determine ifa patient's serum naturally contains the factors which are able tosuppress or activate the growth of tumor cells.

In this procedure, the patient's serum, and/or WBC's, is combined withvarious biological response modifiers (BRM) and co-cultured with a tumorcell line. The percentage of stimulation or percentage of inhibition ofthe tumor cell growth is determined. This information enables theoncologist to determine appropriate and customized treatment for eachpatient.

SPECIMEN REQUIREMENTS AND COLLECTION

Label all specimens with patient name, date drawn, time drawn, andphlebotomist's initial. A unique tracking number will be assigned toeach individual specimen to assure proper specimen tracking.

Requirements:

1. Whole blood (WB)--10 ml

2. Serum--3 ml

3. Bone Marrow (BM)--one BM aspiration in 25 ml of transport media. Theoptimum amount is 3 ml at 1×10⁶ cells per ml.

4. Plasmapheresis specimen--3 ml

Both a whole blood specimen and a serum specimen are required forcomplete immunomodulatory evaluation. However, in the event that onlyone of the two specimens (WB or serum) is available, partialimmunomodulatory testing may be performed.

Collection, Processing, and Handling:

The patient will be properly identified by the nurse or phlebotomist(i.e., check wristband, verify patient name).

Specimens will be aseptically collected by the nurse or a phlebotomist.Gloves and lab coat must be worn while drawing or handling the specimenand "universal precautions" must be observed.

1. Whole Blood

a. Collection--Add 2500 units of preservative free heparin (Calciparine)to a 10 ml red top vacutainer tube. Perform venipuncture and draw thepatient's blood into tube. Label all tubes with patient's name, date andtime collected. Invert several times to mix anticoagulant. Refrigeratetube until ready for separation of the mononuclear layer by the densitygradient procedure (Reference is made to Procedure 4, infra., at partB-2).

Minimum amount whole blood=7 ml

Optimum amount whole blood=10 ml

b. Unacceptable Specimens--Clotted specimens, grossly hemolyzedspecimens, or frozen specimens are unacceptable.

c. Rejection of Specimens--When any criteria are not met, theunacceptable specimens may be tested if necessary.

2. Serum

a. Collection--Use a red top vacutainer tube to perform venipuncture anddraw 10 ml of the patient's blood into the tube. Centrifuge the serumspecimen at 2000 RPM for 10 minutes at 25° C. Refrigerate tube untiltesting. Aseptically draw off serum and place in a separate tube.

Minimum amount serum=3 ml

Optimum amount serum=5 ml

b. Unacceptable Specimens--Specimens with excessive fibrin clotting areunacceptable.

c. Rejection of Specimens--When any criteria are not met, theunacceptable specimens may be tested if necessary.

3. Plasmapheresis

a. Collection--Add 2500 units of preservative free heparin (Calciparine)to a 10 ml red top vacutainer tube. Draw from the plasma collectioncontainer at the beginning of the plasmapheresis procedure. Label tube#1. Refrigerate until testing. Minimum amount of plasma=3 ml

Optimum amount of plasma=5 ml

b. Unacceptable Specimens--Specimens with excessive fibrin clotting areunacceptable.

c. Rejection of Specimens--When any criteria are not met, theunacceptable specimens may be tested if necessary.

4. Bone Marrow

a. Collection--Not more than 15 minutes prior to the BM procedure, add2500 units of Calciparine per 25 ml of tissue transport media usingaseptic techniques. Using aseptic techniques, quickly add the BMspecimens to the transport tube and mix well. Refrigerate the tube untilready for separation of the mononuclear layer by the density gradientprocedure (see part IV, section B-2).

b. Unacceptable Specimens--Grossly clotted specimens, grossly hemolyzedspecimens, or frozen specimens are unacceptable.

c. Rejection of Specimens--When any criteria are not met, theunacceptable specimens may be tested if necessary.

MATERIALS:

The following materials are required for this procedure:

Laminar Flow Hood

Bacto-Agar

Balance

Weighing Paper

Weighing Tools

Capped Bottles (Sterile) (-250 ml, -500 ml)

Distilled Water (Sterile)

Microwave Oven

Autoclave

RPMI 1640 Media

Gentamicin

Disposable Pipettes (Sterile) (-1 ml, -2 ml, -5 ml, -10 ml, -25 ml)

Glutamine

Trace Elements

2-Mercaptoethanol with Dulbecco's PBS

ITS Solution

Fetal Bovine Serum

Test Tubes (Capped) (-5 ml, -10 ml)

Petri Dishes (Sterile) (Regular 100×15 mm,

Gridded 35 mm)

Falcon Tubes

Conical Tube (Sterile-Plastic)

Trypan Blue Stain (0.4˜)

Hemocytometer with Cover Slips

Microscopes (Tissue Culture Inverted Microscope, Light Microscope)

pH Paper (Narrow Spectrum)

Graduated Cylinders (50 ml, 100 ml)

Histopaque-1077

Water Bath

Suction Pipetter

Refrigerated Centrifuge

CO₂ Incubator

Biological Response Modifiers (Recombinant Interferon Alpha 2a,Recombinant Interleukin-2, mAB to Interferon Alpha)

Vortex Mixer

Refrigerator (2-8 C.)

Hand Counter

Sharps Container

Pasteur Pipettes

Dropper Bulbs

-70° C. Freezer

Precise Surface Disinfectant

Micro Glassware Disinfectant/Cleanser

Beaker (250 ml)

Betadine Disinfecting Solution

Syringes (1 cc) (Sterile)

Needles (Various Gauges)

Vacutainer Tubes (Red Tops and "Tiger Tops")

MEDIA AND REAGENT PREPARATION

Prepare Agar:

Lower layer (2.5% working solution): Weigh out 2.50 gm of Bacto-Agar andplace in a 100 ml capped bottle. Add 100 ml distilled water. Swirlgently to mix.

Upper layer (1.5% working solution): Weigh out 1.50 gm of Bacto-Agar andplace in a 100 ml capped bottle. Add 100 ml distilled water. Swirlgently to mix. Place both bottles in the microwave with caps loosened.Microwave for 60 seconds at full power, mixing every 20 seconds. Avoidboiling. The agar will appear clear when ready. If solution boils over,do not use it. Re-make the agar solution. Loosely tape cap withautoclave indicator tape and autoclave both solutions for 20 minutes at22 psi and 250°-270° F. Place both bottles in the water bath under thehood. This bath must be kept at 45°-50° C. at all times. The agar shouldcool down to 50° C. before use. Swirl agar to mix well before using.

Prepare Media:

After preparation of each media, label the final product with thefollowing: 1) Name of Product; 2) Preparation Date; 3) Expiration Date;4) Storage Recommendations; 5) Technologist's Initials

10% RPMI 1640 (1×) G,G fortified:

    ______________________________________                                        AMOUNT         COMPONENT                                                      ______________________________________                                        500.0 ml       RPMI 1640 (1X) with L-Glutamine                                50.0 ml        Fetal Bovine Serum, Qualified, Heat                                           Inactivated                                                    5.0 ml         L-Glutamine (100X) 200 mM                                      2.5 ml         Penicillin/Streptomycin                                        2.0 ml         Trace Element Mix (100X), Lyophilized                          0.5 ml         2-Mercaptoethanol                                              5.0 ml         Insulin-Transferrin-Sodium Selenite, Media                                    Supplement, Lyophilized, Gamma-Irradiated                      ______________________________________                                    

Combine all components under a sterile laminar flow hood. Sterilize byfiltration using a 0.22 micron cellulose acetate membrane filter with a60 micron prefilter. Store the prepared media at 2°-10° C.

10% RPMI 1640 (2×) G,G Fortified:

    ______________________________________                                        AMOUNT       COMPONENT                                                        ______________________________________                                        500.0 ml     RPMI 1640 (2X) Powdered Cell Culture Medium                                   with L-Glutamine (Reconstitute one powder                                     pack with a total of 500.0 ml of sterile                                      distilled water)                                                 50.0 ml      Fetal Bovine Serum, Qualified, Heat                                           Inactivated                                                      5.0 ml       L-Glutamine (100X) 200 mM                                        2.5 ml       Penicillin/Streptomycin                                          2.0 ml       Trace Element Mix (100X), Lyophilized                            0.5 ml       2-Mercaptoethanol                                                5.0 ml       Insulin-Transferrin-Sodium Selenite, Media                                    Supplement, Lyophilized, Gamma-Irradiated                        ______________________________________                                    

Under a sterile laminar flow hood, measure out 450 ml of steriledistilled water and place in a sterile 500 ml reagent bottle. Add thepowdered medium to the water with gentle stirring. Rinse out the insideof the package to remove all traces of powder. Add 1.0 g of NaHCO₃ per500 ml of medium. Dilute with sterile distilled water to 500 ml. Adjustthe pH of the preparation to 0.2-0.3 below the desired final working pH(pH units will usually rise 0.1-0.3 upon filtration); use of 1N NaOH or1N HCl is recommended. After the pH has been adjusted, keep thecontainer closed until the medium is filtered. Sterilize immediately bymembrane filtration using a 0.22 micron cellulose acetate membranefilter with a 60 micron prefilter. Store the prepared media at 2°-10° C.

Prepare Lower Layer Stock Media:

    ______________________________________                                        125 ml         Fetal Bovine Serum                                             125 ml         2 × G, G Fortified Solution                              250 ml         1 × G, G Fortified Solution                              ______________________________________                                    

Mix, label, and store at 2-10° C.

Prepare Upper Layer Stock Media:

    ______________________________________                                        150 ml         Fetal Bovine Serum                                             150 ml         2 × G, G Fortified Solution                              150 ml         1 × G, G Fortified Solution                              ______________________________________                                    

Preparation of Drugs:

Prepare a working concentration for the drugs to be tested. All drugdilutions are made with RPMI 1640 1× G,G Fortified.

To prepare IFN Alpha-2a in a 10 ml capped tube, mix 0.1 ml Roferon(3,000,000 units/ml) with 9.9 ml 1× G,G, Media. Label tube with name ofdrug, preparation date, expiration date (1 week from the day ofpreparation), concentration (30,000 u/ml) and technician's initials.

To prepare IL-2 in 10 ml capped tube: Remove working stock aliquot ofIL-2 (10 units/ml) from refrigerator. If the aliquot is not available inrefrigerator, follow the IL-2 aliquot procedure found in the aliquotprocedure book. The stock tubes are kept in the -48° C. freezer. Thedosage for testing will be 0.1 ml, which will equal 1 unit ofinterleukin-2. Therefore, 1 unit of interleukin-2 will be tested in theimmunomodulatory procedure. Interleukin-2, in this media, can bemaintained for 30 days at 2°-8° C.

To prepare LI-8-ABIFN (mAB to IFN alpha 2A) in a 10 ml capped tube,remove working aliquot (neutralizing effect of 1000 units/ml fromfreezer). The dosage will be 0.1 ml in the assay to achieve aconcentration of 100 units of neutralizing ABIFN.

PROCEDURE:

Lower Layer (2.5% agar):

Use lower layer stock media and dispense 8 ml of stock media into theappropriate number of sterile 15 ml tubes with caps. (Approximately 8plates can be made from 1 tube.) Keep capped at room temperature untilneeded to prepare lower layer plates or store capped overnight at 2°-8°C. for use the following day. Warm to room temperature before use. Whenready to prepare the lower layer plates work quickly to preventpremature solidification of the agar. Using a sterile 10 ml pipet drawup 2.0 ml of the 2.5% agar mixture. Dispense the 2.0 ml into one of the15.0 ml plastic tubes containing 8 ml of lower layer media, and mixtwice by drawing up and dispensing into the tube. Draw up 9.0 ml of themixture and dispense 1.0 ml into each 35 mm gridded plate. Swirl tocover the bottom of the plate. Take care to prevent bubbles. Prepareonly 8 plates at a time so that the agar does not solidify before it isplated. Allow plates to sit undisturbed on a flat surface until the agarhas solidified. Discard any plates with uneven agar distribution. Storeplates at room temperature for same day use or overnight in a 37° C. CO2 incubator for use the following day.

Preparing the Cells Lines:

Generally, L929 cells are used in the assay, but colon, prostate, breastor ovarian cells may also be used.

For L929 cells, microscopically examine the liquid culture flasks of thedesired cell line and determine by confluency which flasks to use.Remove supernatant with pipet and discard. Add 5 ml Trypsin. Expose tocell layer and remove 2 ml. Incubate flasks with Trypsin for minute. Hitflask with the palm of your hand to knock cells off. Add 7 ml of L929cell media to stop reaction. Transfer cells to a sterile 50 ml conicaltube, add more media (to bring to 20 ml). Centrifuge tube @ 1600 RPM for5 minutes with brake on 2. Remove supernatant and resuspend pellet inL929 cell media. Centrifuge cells at 1600 RPM for 5 minutes with brakeon 2. Remove supernatant, resuspend pellet in appropriate media.

Preparing the Patient Cells (from whole blood with Calciparine, or BMSpecimen with Calciparine):

Into an appropriate size (depends on sample volume) capped plastic tubeslabeled with each patient's name, dispense the appropriate volume ofhistopague (use equal amounts of histopaque to equal amounts of wholeblood, i.e., if a whole blood tube containing 8 ml of blood is received,dispense 4 ml of histopaque and 4 ml of whole blood into each of two 15ml capped tubes. If 75 ml of BM is received, dispense approximately18.75 of histopaque and 18.75 of BM into four 50 ml conical tubes. Layerin the whole blood very slowly at a 45° C. angle on the histopaque.Centrifuge at 1600 RPM for 20 minutes in centrifuge at 4° C., brake on2. Remove and discard the supernatant. Remove the cell interface andplace in a separate tube containing 10˜ RPMI 1× G,G media. Dilute thecells with media quickly because the histopaque is very toxic to thecells if left on them for extended periods of time. Wash cells twicewith 10˜ RPMI 1× G,G media. Centrifuge at 1600 RPM for 5 minutes withthe brake on 2. Remove and discard the supernatant. Resuspend the cellpellet in 5 ml RPMI 1× G,G media.

PERFORM CELL COUNT:

In a 12×75 ml glass tube, combine: 0.7 Media; 0.2; Trypan Blue; and 0.1Cell Suspension. This is a 1:10 Dilution. Mix well. Charge one chamberof the hemocytometer with the cell suspension. Under the lightmicroscope, count the live cells (those which have not absorbed TrypanBlue) in the 4 corner 1 mm² squares. The concentration of cell lines andpatient cells for the IM assay will be as follows (cells per ml):

L-Cells5×10⁵

Colon1×10⁵

Prostate5×10⁴

Breast5×10⁴

Ovarian2×10⁴

Patient1×10⁶

Count the total number of cells (stained and unstained) in the 4 corner1 mm² squares.

CALCULATION FOR CELL COUNT:

Use the following cell count formula:

    Cells per ml=average # cells×dilution factor×hemocytometer factor.

EXAMPLE CALCULATION:

Patient cells are brought up in 10 ml of media. 0.1 ml of cells areadded to 0.2 ml of Trypan Blue and 0.7 ml of media. 4-1 mm² squares werecounted and cell count=80 cells.

    20 cells (80 cells 4-1 mm.sup.2 squares=average # of cells)×10 (dilution factor)×10.sup.4 =2×10.sup.6 cells/ml.

Calculate % viability using the following formula:

    % of viable cells×100=% viability total # of cells

Adjust volume to amount needed for the appropriate cell concentrationfor the assay:

    (V.sub.1 C.sub.1 =V.sub.2 C.sub.2)

V₁ =10 ml V₂ =?

C₁ =2×10⁶ cells/ml C₂ =1×10⁶ cells/ml ##EQU4## 20 ml=V₂ 20 ml-10 ml=10ml

Ten ml of media must be added to have a volume of 1×10⁶ cells/ml.

Label the appropriate number of plates with the followinginformation: 1) Specimen number; 2) Patient name; 3) Name of BRM; 4)Setup date.

Preparing Upper Layer:

Label and arrange 15 ml sterile capped tubes for each control, patientserum, patient whole blood sample, that you need in a test tube rackaccording to Table 2 (infra.). Be sure to include a cell control, and acontrol for each BRM to be tested. The cell control should containeverything the others contain except for BRM. A separate set of controltubes must be set up for each cell line assayed (colon, L cell,prostate). Dispense the upper layer stock media into each tube, then addappropriate amounts of BRM, ABIFN, cell line and serum or patient wholeblood. See Table 2 as follows:

                                      TABLE 2                                     __________________________________________________________________________    FOLLOW THIS TABLE FOR UPPER LAYER VOLUMES                                     (ALL IN ml):                                                                                        PTWBC                                                           UPPER         OR   L    AGAR                                                                              TOTAL                                             LAYER                                                                              BRM ABIFN                                                                              SERUM                                                                              CELLS                                                                              1.5%                                                                              VOLUME                                    __________________________________________________________________________    CELL    2.1  --  --   --   0.3  0.6 3.0 ml                                    CONTROL                                                                       IFN     2.0  0.1 --   --   0.3  0.6 3.0 ml                                    CONTROL                                                                       IL2     2.0  0.1 --   --   0.3  0.6 3.0 ml                                    CONTROL                                                                       ABIFN                                                                         CONTROL 2.0  --  0.1  --   0.3  0.6 3.0 ml                                    PT. SERUM                                                                             1.9  --  --   0.2  0.3  0.6 3.0 ml                                    PT. SERUM/                                                                            1.8  --  0.1  0.2  0.3  0.6 3.0 ml                                    ABIFN                                                                         PT. SERUM/                                                                            1.8  0.1 --   0.2  0.3  0.6 3.0 ml                                    IFN                                                                           PT. SERUM/                                                                            1.8  0.1 --   0.2  0.3  0.6 3.0 ml                                    IL2                                                                           PT. WBC 1.9  --  --   0.2  0.3  0.6 3.0 ml                                    PT. WBC/                                                                              1.7  --  --   0.2  0.3  0.6 3.0 ml                                    SERUM                 0.2                                                     PT. WBC/                                                                              1.6  --  0.1  0.2  0.3  0.6 3.0 ml                                    SERUM/ABIF            0.2                                                     PT. WBC/                                                                              1.8  0.1 --   0.2  0.3  0.6 3.0 ml                                    IFN                                                                           PT. WBC/                                                                              1.6  0.1 --   0.2  0.3  0.6 3.0 ml                                    IFN/SERUM             0.2                                                     PT. WBC/                                                                              1.8  0.1 --   0.2  0.3  0.6 3.0 ml                                    IL-2                                                                          PT. WBC/                                                                              1.6  0.1 --   0.2  0.3  0.6 3.0 ml                                    IL-2/SERUM            0.2                                                     __________________________________________________________________________

Add all components except agar to the appropriate tube. For thefollowing, one tube should be finished before moving on to the next.With a 2.0 ml pipet, draw up 0.6 ml of 1.5% agar. Dispense the agar intothe tubes one at a time, starting with tube #1. Mix by aspirating anddispensing twice. Draw up 2.2 ml of solution and dispense 1.0 ml intoeach gridded petri plate (on top of the other layer) taking care not toproduce bubbles in the agar. Gently swirl plate to allow for evendistribution. Allow agar to solidify, and then add distilled water(approximately 0.5 ml) to humidity plate and place in large labeledpetri plate. Repeat steps 5-7 for each plate to be set up. Incubate thecultures at 37° C. in a humid, 5% CO₂ -enriched atmosphere.

REPORTING RESULTS:

Reading semi-solid culture plates:

Read the plates after 7 days as follows: Scan the plate for colonies andaggregates. If they are present, count all the squares to obtain thenumber of aggregates and colonies in the plate (aggregates=4-20 cellsper cluster) (colony is >20 cells per cluster). If no colonies oraggregates are present, count all individual cells (cell line andpatient's WBC's) in 4 squares and take the average. Multiply the averageby 15×13.36 (15=# of square in the center row) (13.36=area of theplate). Plates may be read at 5, 6 or 8 days. Record all data inscientific notation on the report form.

Procedure 4 Human Interferon Assay

Interferons are cytokines that have the ability to inhibit the growth ofviruses and to protect infected cells against viral cytopathic effects.The immunoregulatory functions of interferons such as the increase innatural killer (NK) lymphocyte activity, the increase inhistocompatibility antigens, the activation of monocyte/macrophages, andB-cell function have also proven to be of clinical importance. The firstnatural interferon was discovered by Isaac and Linderman in 1975, andrecombinant interferon alpha 2 was registered by the FDA in 1986ushering in a new phase of biotherapy. The goal of this assay is todetermine the level of interferon in international units/ml in patient'sserum. Potency of the human interferon of serum samples and controlswill be determined using WISH cells challenged with Vesicular StomatitisVirus measuring the cytopathic effect.

SPECIMEN REQUIREMENTS, COLLECTION AND HANDLING

Specimens are labeled with the following information: 1) Patient name;2) Date drawn; 3) Time drawn; 4) Phlebotomist's initials; 5) Test name.

Serum-Amount required:

    Optimum=3.0 ml

    Minimum=1.0 ml

The specimen must be aseptically collected in a "tiger" top vacutainertube, allowed to clot, then centrifuged at room temperature for 10minutes at 2000 rpm. The serum should be poured over into a sterileplastic tube, capped, appropriately labeled, and stored frozen until theassay is performed.

Plasmapheresis--Amount required:

    Optimum=3.0 ml

    Minimum=1.0 ml

The specimen must be collected using sterile technique into a 10 ml redtop vacutainer tube from the plasma bag obtained during theplasmapheresis procedure. Add 2500 units of calciparine to the 10 ml redtop vacutainer tube containing the plasmapheresis sample and mix well.In addition to the information required above, the specimen should belabeled with the bag number from which it was obtained, i.e. #1, #2,etc. The specimen must be poured over into a sterile plastic tube,capped, appropriately labeled, and stored frozen until the assay isperformed.

EQUIPMENT:

The following equipment is needed for this procedure:

Sterile laminar flow hood with ultra-violet light

Freezer -70° C.

Refrigerator 2°-8° C.

Autoclave

Analytical balance

Refrigerated centrifuge

CO₂ incubator

Spectrophotometer (Bio-Rad 96-well plate reader with 540 nm filter)

96-well microtiter plates--sterile flat bottom with covers Falcon 3872

Pipet aid

Micropipettes

Costar octapettes--50 ul, 100 ul, & 200 ul

Wheaton multi-channel pipet--50-200 ul range

MLA-40 ul

Sterile disposable:

Pipettes (1 ml, 5 ml, 10 ml, and 25 ml)

Pipet tips Wheaton 851247

Plastic troughs

Gauze

Cryovials

50 ml conical tubes with caps

Test tubes

Culture flasks (75 cm and 150 cm)

Kimtex wipers

Biohazardous waste can and bags

Stainless steel pan with cover

MATERIALS:

The following materials are needed for this procedure:

Basal Medium Eagle 1× (BME), 500 ml Gibco Cat. No. 320-1010AJ

Fetal Bovine Serum, 100 ml Hyclone Cat. No.

A-1111-D

L-Glutamine, Cat. No. 320-5030AG

Hepes Buffer Solution (1M), 100 ml Gibco Cat. No. 380-5630AG

Penicillin/Streptomycin

WISH cells (Human Amnion) ATCC 25-CCL

Vesicular Stomatitis Virus ATCC

Human Interferon (alpha) Reference NIAID Cat. No. Ga23-902-530

Gamma Interferon control

Neutral red dye Sigma No. N-2880

Phosphate buffered saline (PBS)

PBS without Ca++ and Mg++, Ph 7.4 Gibco

Cat. No. 310-4190AJ

PBS with Ca++ and Mg++, Ph 6.8 Gibco

Cat. No. 310-4040AJ

Glacial acetic acid Sigma Cat. No. A-6283

Distilled water

Ethyl Alcohol Aldrich Cat. No. 18,738-0

Patient sera

One-Stroke Environ Calgon Vestal No. 539708

Trypsin-EDTA 1×, 100 ml Gibco Cat. No. 610-520OAG

Trypan Blue Gibco 6305250AG

REAGENT PREPARATION:

All media must be filtered for sterility, labeled with name, preparationand expiration dates, storage information, and technician's initials.Prepared media is stable for three months at 2°-8° C.

BME with 15% FBS:

Add 75 ml of FBS, 5.0 ml Pen/Strep 10 ml glutamine, and 5 ml hepes to a500 ml bottle of BME. (For feeding WISH cells.)

BME with 10% FBS:

Add 50 ml of fetal bovine serum, 2.5 ml Pen/Strep, 10 ml glutamine, and5 ml hepes to a 500 ml bottle of BME.

BME with 2% FBS:

Add 10 ml of FBS, 2.5 ml Pen/Strep, 10 ml glutamine, and 5 ml hepes to a500 ml bottle of BME.

Vesicular Stomatitis Virus (VSV):

Prepare two 150 cm² culture flasks of WISH cells. Discard the media whenthe cells become confluent. Wash once with fresh BME with 2% FBS. Dilutea VSV stock preparation 1000 times with BME with 2% FBS. Inoculate 8-10ml per flask of the 1:1000 VSV into each WISH cell flask such that itcovers the entire cell layer. Incubate the flasks for 1 hour at 37° C.in a 5% CO2 incubator. Add 15 ml of the BME with 2% FBS to each flaskand incubate at 37° C. in a 5% CO2 incubator for 1-3 days or until celllayers show nearly complete viral cytopathic effect (CPE). Harvest theculture fluid and clarify by centrifugation at 3000 RPM for 20 minutesat 4° C. Aliquot the virus containing supernatant into 10 ml tubes andstore at -70° C. This virus stock usually contains 1×10.sup. 9plaque-forming units/ml when its infectivity is quantitated in WISHcells. Dilute one 10 ml stock VSV tube 1:10 with BME with 2% FBS toyield a working VSV of 1×10⁸ plaque forming units/ml. Aliquot 1 ml ofworking VSV into cryovials and store at -70° C. Dilutions of 1:100,1:200, and 1:300 of working VSV should be made and inoculated ontoconfluent WISH cells in a 96-well plate to determine the dilution thatyields nearly 100% CPE in 24-48 hours.

Human Alpha Interferon (National Institute of Health: catalog#Ga23-902-530):

Reconstitute with 1.0 ml of sterile distilled water being careful toavoid any loss of material in the neck of the ampule. Dilute to aconcentration of 105 IU/ml of interferon with BME with 2% FBS (noPen/Strep and no hepes). Aliquot 200 ul into cryovials and store in -70°C. freezer. The aliquots are stable for two years at this temperature.

Neutral Red Dye:

Prepare 0.1% stock solution of Neutral Red Dye by adding 500 mg of reddye powder to 500 ml of distilled water. Mix with magnetic stir bar atroom temperature for 1 hour. Autoclave for 10 minutes at 15 psi. Cool.Label and store stock Neutral Red Dye in a brown bottle and refrigerate.Staining Solution--prepare a 15% staining solution by adding 85 ml PBSwith Ca⁺ + and MG⁺ + (Ph 6.85) to 15 ml stock Neutral Red Dye.

Eluting Solution:

To 1 ml glacial acetic acid add 49 ml distilled water and 50 ml 100%ethanol. Label and store in glass capped bottle at room temperature.

Gamma Interferon Control:

Obtain Actimmune (3.0×10⁶ units/ml). Dilute 0.05 ml (50 ul) of Actimmunewith 49.95 ml of BME with 2% FBS and mix well to obtain a concentrationof 3000 units/ml. Aliquot 10 ml into each of 5 sterile capped tubes andlabel with "stock IFN gamma," 3000 units/ml, preparation and expirationdates. (Stable for 2 years at -70° C.). Dilute 1 ml of stock IFN gammawith 9.0 ml of BME with 2% BME for a working concentration of 300units/ml. Aliquot 200 ul of this concentration into cryovials, label,and store at -40° C. (Stable for 2 years.). Run in parallel with currentcontrol to obtain range prior to putting into use.

PROCEDURE:

Day One:

Generate a worksheet from the interferon data base for 24 samples.Number the worksheet with #1 as the reference, patient samples #2-#25,the gamma control #26, and the alpha control #27. Retrieve the patientsamples, interferon reference and controls from the freezer. Verifysamples with the worksheet. Label nine sterile 96-well microtiter plateswith sample numbers (run in duplicate), plate number and date. Two 150cm² flasks of confluent WISH cells are harvested and washed by theliquid culture technician. The tube of cells is labeled with the name ofthe cells, the passage number, date, and technician's initials. Note:The passage of WISH cells should be less than P250. Before this passageis reached, cells of younger passage should be retrieved from liquidnitrogen and started in liquid culture.

Perform the cell count in the following manner: in a 12×75 ml tube,combine 0.5 ml of WISH cells and 0.5 ml of 0.4% Trypan Blue (1:2dilution). Mix well and charge the hemacytometer chamber. Under thelight microscope, count the viable WISH cells in the four corner 1 mm²squares and divide by 4 to obtain the average for 1 square. Count thetotal number of cells in the four corner 1 mm² squares and divide by 4to obtain the average for 1 square. Determine the % viability bydividing the viable cell count by the total cell count and multiplyingby 100. Record the % viability on the cell count worksheet.

Determine the number of cells/ml by using the following equation:

    Avg. # of viable cells/square×dilution factor×hemacytometer factor=cells/ml

Dilute the WISH cells to 3.5×10⁵ cells/ml according to the followingformula:

    V.sub.1 C.sub.1 =V.sub.2 C.sub.2

For example:

Volume₁ =40 ml Concentration₁ =2.0×10⁶

Volume₂ =? Concentration₂ =3.5×10⁵ ##EQU5## V₂ =229 ml

Add 189 ml of BME with 10% FBS to the original volume of 40 ml to obtain3.5×10⁵ cells/ml. Set aside diluted WISH cells until ready to be addedto the microtiter plates. Using sterile techniques, add 100 ul of 10%BME to each well (including blanks) of the 96-well microtiter plates. Tothe wells in column #1, B-G, add an additional 60 ul of 10% BME. To wellpositions B1 and C1 of plate #1 (each sample will be run in duplicate)add 40 ul of the reference. To well positions D1 and E1 add 40 ul ofpatient serum. To each successive pair of wells, add 40 ul of theappropriate patient serum. In addition to the patient samples, set up agamma interferon control and an alpha interferon control (reference) onthe last plate of the assay. Make serial dilutions with the 100 uloctapet from column 1 to column 10 and rows B-G by mixing up and downseveral times, then carrying 100 ul to the next column through the 10thcolumn. Discard the remaining 100 ul from the 10th column. Add 100 ul ofthe 3.5×10⁵ cells/ml WISH cell suspension to all wells except blankwells in rows A and H. Designate column 11 as cell control and column 12as viral control on each plate. Incubate plates 18-24 hours at 37° C. at5% CO₂. Lay out the stainless steel pan containing OneStroke Environ,diapers and gauze squares in hood with white light overnight.

Day Two:

Check plates under the inverted microscope for confluency. Check fortoxicity of patient sera (black edges). Using sterile techniques, dumpthe media from the plates into the stainless steel pan containingOneStroke Environ, blot on gauze. Wash the cells by adding 100 ul of BMEwith 2% FBS to all the wells except the blank wells, dump, and blot. Add50 ul of 2% BME to column 11. Add 100 ul of 2% BME to all wells(including blanks). Add 50 ul VSV (in a concentration that yields 90%CPE in the viral control in 24-48 hours) to all the sample wells incolumns 1-10 and column 12. Incubate 24-48 hours at 37° C. with 5% CO₂.

Day Three:

Check plates under the microscope for 90-100% CPE in the viral control.Look for 50% CPE in the reference and samples. The 50% CPE should fallbetween columns 4 and 6 for the reference. Incubate plates until this isachieved. Place the stainless steel pan containing One-Stroke Environalong with Kimtex wipers sufficient for blotting the plates under thehood. Dump plates and blot. Add 100 ul of 15% neutral red dye to all thesample wells. Incubate at 37° C. with 5% CO₂ for 45 minutes. Dump platesand blot. Rinse with 200 ul PBS at pH 6.85. Dump and blot. Plates may beleft to dry inverted on Kimtex wipers on a tray for several days beforeeluting. Elute by adding 100 ul of the eluting solution to each samplewell.

Read on the microplate reader with a 540 nm filter. Select Microman fromthe menu to access the Microplate Manager program. Under Analysis,select Multiple Readings.

Select:

Single Wavelength

540 nm

Mixing duration--5 seconds

Reading per plate--1

Number of plates--9

Filename--date (i.e., 0722)

Remove cover of microtiter plate #1 and place in the reader. SelectStart. After all the plates have been read, print the raw data reportsas follows: Under file, select Open and Lotus Results. Arrow down tofilename, or type in the file name and plate number. Select Open. Ifmessage appears "Save current results . . . ," say NO. When Raw DataReport appears on the screen, under File, select Print. Select Raw DataReport and OK. Under file select Close. Repeat for each plate to beprinted.

RESULTS:

A. Average the optical density (O.D.) values for the cell controls(column 11) and viral controls (column 12).

B. Average the O.D. values for the duplicates of the patients andreference samples--i.e. average B & C for each column 1-10, average D &E for each column 1-10, etc.

C. To determine the O.D. at 50%: ##EQU6##

D. To enter the O.D. values and their corresponding IFN dilutions:

1. Enter the 1st average O.D., press X-Y

2. Enter corresponding 1st IFN dilution, press LOG, then Ε+

3. Enter the 2nd average O.D., press X-Y

4. Enter corresponding 2nd IFN dilution, press LOG, then Ε+

5. Enter the 3rd average O.D., press X-Y

6. Enter corresponding 3rd IFN dilution, press LOG, then Ε+

E. To determine the correlation coefficient (r):

1.Press 2nd, then CORR (÷)

2. The correlation coefficient (r) should be -1.0±0.1.

3. Enter O.D. 50%, press 2nd, then y' (x)

F. To convert the titer (log 10) to units/ml, press INV, then LOG

G. To clear data, press 2nd, then STO, then CE/C

H. To convert IFN units/ml to International Reference Units/ml:

1. IFN units/ml of Reference=Factor Int'l Ref. Units/ml of Reference

2. Sample IFN units/ml=Sample titer (IRU/ml) Factor

Procedure 5 Human Tumor Colony-Forming Chemosensitivity Assay(Pharmacosensitivity)

The Human Tumor Colony Assay (HTCA: clonogenic or tumor stem cell assay)is an in vitro culture system employing semi-solid medium supportoriginally described by Salmon and Hamburger, et al. Using HTCA, thegrowth and chemosensitivity of clonogenic tumor cells present in freshbiopsy specimens of human tumors can be investigated. Since thistechnique was first described, there has been a marked increase in thedirect study of human tumors in vitro. Excellent evidence has beenobtained which establishes that colonies grown in HTCA are comprised oftumor cells and that clonogenic cells within tumor colonies have theproperty of self-renewal (the defining property of a tumor stem cell).Chemosensitivity testing with specific agents in HTCA has documentedstriking degrees of heterogeneity in drug sensitivity from patient topatient, even for tumors of the same histopathology. Clinicalcorrelations have been made between in vitro chemosensitivity and theresponse of patients with metastatic cancer to chemotherapy. In a seriesof trials, HTCA has had a 71% true-positive rate and a 91˜ true-positiverate for predicting the drug sensitivity and resistance, respectively,of cancer patients to specific chemotherapeutic agents. The assay thusappears to be a prognostic factor which identifies chemosensitivepatients and which may allow some individualization of chemotherapy.

Tumor specimens, either from solid tumor masses, malignant ascites, orbone marrow are mechanically disaggregated into a suspension which is asclose to a "single-cell suspension˜as possible. These cells along withchemotherapeutic agents, biological response modifiers, and hormones aresuspended in an agar-containing culture medium and then layered or"plated" onto a semi-solid underlayer. The underlayer prevents normalhuman fibroblasts, a major tumor stromal component, from adhering to theculture dish bottom and forming colonies which might be confused withcolonies of tumor-cell origin. After 7 days, 14 days, and 21 days ofincubation in a humid, CO2-enriched atmosphere at 37° C., colonies(greater than 20 cells) and aggregates (4-20 cells) are counted. Anegative control and a positive control are set up for each tumor. Fromthis data the effect of each specific drug on the tumor can bedetermined.

SPECIMEN REQUIREMENTS, COLLECTION AND HANDLING:

This assay may be performed on the following specimen types:

Solid tumors obtained during surgery or biopsy

Bone marrow aspirates and biopsies

Ascites

Pleural fluids

Thoracentesis fluids

The sample should be sufficient to yield an optimum of 20 ml of 1.0×10⁶viable cells/ml. A minimum of 12 ml of 1.0×10⁶ viable cells/ml isacceptable. All specimens should be labeled with patient name,collection date, tumor type, and initials of technician.

MATERIALS:

The following materials are needed for this procedure:

Laminar Flow Hood

Bacto-Agar

Analytical Balance

Weighing Paper

Weighing Tools

Sterile Capped Bottles (100 ml, 250 ml, 500 ml; and 1,000

Distilled Water

Microwave Oven

Autoclave

RPMI 1640 Media (1× and 2×)

HCL (1N)

Sodium Bicarbonate

Penicillin/Streptomycin

Disposable Pipettes (sterile) (1 ml; 2 ml; 5 ml; 10 ml, 25 ml)

Select-A-Pette Pipetter (1 ml)

Select-A-Pette Tips (sterile)

Eppendorf Pipettes

Eppendorf Combitips (sterile) (2.5 ml; 5.0 ml; 12.5 ml)

Glutamine

Trace Elements

2-Mercaptoethanol With Dulbecco's PBS

ITS Solution

Fetal Bovine Serum

Test Tubes (12×75 Glass-disposable; 15 ml Capped-sterile and disposable)

Petri Dishes (sterile) (Routine-100×15 mm; Gridded Plates-35×10 mm)

Sterile Capped 50 ml Conical Tubes (Falcon)

Homogenizer

Trypan Blue Stain (0.4˜)

Hemocytometer With Cover Slips

Microscopes (Inverted; Light)

Hand Tally Counter

Ph Paper (narrow spectrum)

Graduated Cylinders (50 ml and 100 ml)

Histopaque-1077

Water Bath

Pipet Suction Aide

Refrigerated Centrifuge

Liquid Culture Flasks (25 cm²)

CO₂ Incubator

Chemotherapeutic Drugs

Biological Response Modifiers

Hormones

Vortex Mixer

Refrigerator (2°-10° C.)

Scalpel (disposable and sterile)

Sharps Container Pasteur Pipettes 53/4" (disposable)

Dropper Bulbs

-70° C. Freezer

Precise Surface Disinfectant

70% Alcohol In Squeeze Bottle

Contrad Glassware Disinfectant/Cleanser

Thermometer (-20° C.-120° C.)

Beaker (500 ml)

Betadine Disinfecting Solution

Syringes (disposable and sterile) (1 cc; 10 cc)

Needles (various gauges)

MEDIA AND REAGENT PREPARATION:

Agar:

Lower layer=2.5% working solution: Measure out 2.5 gm of Bacto-Agar andplace in a 100 ml capped bottle. Add 100 ml distilled water. Swirlgently to mix.

Upper layer=1.5% working solution: Measure out 1.50 gm of Bacto-Agar andplace in a 100 ml capped bottle. Add 100 ml distilled water. Swirlgently to mix.

Place both bottles in the microwave with caps loosened. Microwave for 60seconds at full power, mixing every 20 seconds. Avoid boiling. The agarwill appear clear when ready. If the solutions boil over, do not use,remake the agar. Place a piece of autoclave tape on each of the looselycapped bottles and autoclave the solutions for 20 minutes at 22 psi.Place both bottles in the water bath under the hood. THIS BATH MUST BEKEPT AT 45°-50° C. AT ALL TIMES. The agar should cool down to 50° C.before use. Swirl agar to mix well before using. After preparation ofeach media, label the final product according to procedures previouslydescribed.

MEDIA:

10% RPMI 1640 (1×):

    ______________________________________                                        AMOUNT         COMPONENT                                                      ______________________________________                                        500.0 ml       RPMI 1640 (1X) with L-Glutamine                                50.0 ml        Fetal Bovine Serum, Qualified, Heat                            Inactivated                                                                   5.0 ml         L-Glutamine (100X) 200 mM                                      1.5 ml         Penicillin/Streptomycin                                        2.0 ml         Trace Element Mix (100X) Lyophilized                           0.5 ml         2-Mercaptoethanol                                              5.0 ml         Insulin-Transferrin-Sodium Selenite                                           Media Supplement, Lophilized, Gamma-                                          Irradiated                                                     ______________________________________                                    

Combine all components under a sterile laminar flow hood. Sterilize byfiltration using a 0.22 micron membrane filter with a 60 micronprefilter. Label and store the prepared media at 2°-10° C.

10% RMPI 1640 (2×) G,G Fortified:

    ______________________________________                                        AMOUNT         COMPONENT                                                      ______________________________________                                        500.0 ml       RPMI 1640 (2X) Powdered Cell Culture                                          Medium With L-Glutamine (Reconstitute                                         one powder pack with a total of 500.0 ml                                      of sterile DISTILLED WATER)                                    50.0 ml        Fetal Bovine Serum, Qualified, Heat                                           Inactivated                                                    5.0 ml         L-Glutamine (100X) 200 mM                                      2.5 ml         Penicillin/Streptomycin                                        2.0 ml         Trace Element Mix (100X) Lyophilized                           0.5 ml         2-Mercaptoethanol                                              5.0 ml         Insulin-Transferrin-Sodium Selenite                                           Media Supplement; Lophilized; Gamma-                                          Irradiated                                                     ______________________________________                                    

Under a sterile laminar flow hood, measure out 450 ml of steriledistilled water and place in a sterile 500 ml reagent bottle. Add thepowdered medium to the water with gentle stirring. Rinse out the insideof the package to remove all traces of powder. Add 1.0 G OF NaHCO₃ per.500 ml of medium. Dilute with sterile DH₂ O to 500 ml volume. Adjust thepH of the preparation to 0.2-0.3 below the desired final working pH of7.2 (pH units will usually rise 0.1-0.3 upon filtration); use of 1N NaOHor 1N HCl is recommended. After the Ph has been adjusted, keep thecontainer closed until the medium is filtered. Sterilize immediately bymembrane filtration using a 0.22 micron membrane filter with a 60 micronprefilter (positive pressure is recommended). Label and store theprepared media at 2°-10° C.

Lower Layer Stock Media:

125 mlFetal Bovine Serum

125 ml2×G,G Fortified Solution

250 ml1×G,G Fortified Solution

Mix, label, and store at 2°-10° C.

Upper Layer Stock Media:

150 mlFetal Bovine Serum

150 ml2×G,G Fortified Solution

150 ml1×G,G Fortified Solution

Mix, label, and store at 2°-10° C.

Preparation of Drugs:

Working aliquots of each drug, hormone, and biological response modifierare prepared every two weeks according to the aliquot procedure. Theworking aliquots are stored at 4° C. and are ready for use.

PROCEDURE:

Prepare the Lower Layer Tubes:

Pipet 8 ml of lower layer stock media into 15 ml sterile capped tubes.(NOTE: Determine the number of plates needed by multiplying the numberof drugs on the testing panel ×2. One lower layer tube yieldsapproximately 8 lower layer plates.) Keep capped at room temperatureuntil needed to prepare lower layer plates or store capped overnight at28° C. for use the following day. Be sure to warm to room temperaturebefore use.

Plate the lower layer plates as follows (WORK QUICKLY TO PREVENTPREMATURE SOLIDIFICATION OF THE AGAR): With a sterile 10 ml pipet, drawup 2.0 ml of the 2.5% agar mixture. Dispense the 2.0 ml of agar into oneof the 15.0 ml plastic tubes containing 8 ml of lower layer media andmix twice by drawing up and dispensing into the tube. Draw up 9.0 ml ofthe mixture and dispense 1.0 ml into each of eight sterile 35 mm griddedplates. Swirl to cover the bottom of the plate. Take care to preventbubbles. Allow plates to sit undisturbed on a flat surface until theagar has solidified. Discard any plates with uneven agar distribution.Store plates at room temperature for same day use or store overnight lna 37° C. CO2 incubator for use the following day.

Prepare the Cell Suspension:

Solid tumor: In a sterile petri dish, using sterile scalpel and forceps,cut the tumor specimen into pea-size portions. Save the originaltransport media for centrifugation. Place the specimen in a glassconical tube that has a matched loose-fitting homogenizer. Add 5.0 ml oftransport media. Gently homogenize the tumor pieces a small portion at atime and transfer the cell suspension to a sterile 50 ml conical tube.When all of the tumor has been homogenized, mix well and then allow cellsuspension to settle, undisturbed, for one minute (this will allowclumps of tissue to settle out). Pipet the supernatant into a steriletube, dilute with RPMI 1× with 10% FBS and centrifuge at 1600 RPM for 5minutes. Remove the supernatant, resuspend the cells in RPMI 1× with 10%FBS, and perform cell count and viability.

Liquid tumor (ascites, bone marrow, etc.): Using a sterile pipet,dispense 20-25 ml of histopaque into the appropriate number of cappedsterile 50 ml conical tubes. Using another sterile pipet, gently layerthe ascites, bone marrow or cell suspension at a 45° C. angle onto thehistopaque using a 1:1 ratio of histopaque to specimen. Centrifuge at1600 RPM for 20 minutes in the refrigerated centrifuge at 4° C. (brakeon 2). Remove and discard the supernatant. Remove the cell interface andplace in RPMI (1×) GG fortified in a pre-labeled tube. DILUTE THE CELLSWITH THE MEDIA QUICKLY BECAUSE THE HISTOPAQUE IS VERY TOXIC TO THE CELLSIF LEFT ON THEM FOR EXTENDED PERIODS OF TIME. Wash cells twice with RPMI1640 (1×) media and centrifuge at 1600 RPM for 5 minutes with a brake of2. Remove and discard the supernatant. Resuspend the cell pellet in 5 mlof RPMI 1× medium.

Perform Cell Count:

In a 12×75 ml glass tube, combine:

0.1 ml of well-mixed cell suspension

0.2 ml of 0.4% Trypan Blue stain

0.7 ml of media

This is a 1:10 Dilution.

Mix well and charge one chamber of the hemocytometer with the cellsuspension. Under the light microscope, count the viable tumor cells(those which have not absorbed the Trypan Blue) in the four corner 1 mm²squares. Count the total number of tumor cells (stained and unstained)in the four corner 1 mm² squares.

Calculation for Cell Concentration:

Perform the viable cell count using the following equation:

    Average # of viable cells per square×dilution factor×hemacytometer factor

Sample calculation:

    20 Cells (average # of viable cells per square)×10.sup.4 ×10 (dilution factor)=2×10.sup.6 cells/ml.

Determine the % viability using the following formula: ##EQU7##

Adjust volume to amount needed for the appropriate cell concentrationfor the assay (this assay requires 1×10⁶ cells/ml) using the followingformula:

    (V.sub.1 C.sub.1 =V.sub.2 C.sub.2)

For example:

V₁ =10 ml V₂ =?

C₁ =2×10⁶ cells/ml C₂ =1×10⁶ cells/ml ##EQU8## 20ml=V₂ 20 ml-10 ml=10 ml

Ten ml of media are needed to have a volume of 1×10⁶ cells/ml.

Prepare Upper Layer Tubes:

Pull and arrange the prepared drugs that are to be tested against thetumor cells. Label the appropriate number of 15 ml capped tubes with thenames of the drugs. Arrange the tubes in a test tube rack. Use the Table3 as follows for upper layer volumes (all in ml):

                  TABLE 1                                                         ______________________________________                                               UPPER LAYER                  AGAR                                             MEDIA      CELLS    DRUGS    1.5%                                      ______________________________________                                        CONTROL  2.1          0.3      --     0.6                                     ABRIN    1.8          0.3      0.3    0.6                                     DRUG #1  1.8          0.3      0.3    0.6                                     DRUG #2  1.8          0.3      0.3    0.6                                     DRUG #3  1.8          0.3      0.3    0.6                                     ______________________________________                                    

Add all components except agar to the appropriate tubes. For thefollowing steps, one tube should be finished before moving on to thenext. With a 2.0 ml pipet, draw up 0.6 ml of 1.5% agar. Dispense theagar into the tube, one at a time, starting with tube #1. Mix byaspirating and dispensing twice. Draw up 2.2 ml of the solution anddispense 1.0 ml into each of two gridded plates (on top of the otherlayer) taking care not to produce bubbles in the agar. Gently swirlplate to allow even distribution. Allow agar to solidify undisturbed,and then add approximately 0.5 ml of distilled water to a third "halfplate" in the large petri dish labeled with the corresponding drug.Repeat steps 5-7 for each drug to be tested. Incubate the cultures at37° C. in a humid, 5% CO₂ enriched atmosphere.

Liquid Culture:

With the remaining cell suspension liquid cultures as follows: Label two25 cm² liquid culture flasks. Add RPMI 1640 1× Fortified plus FBS andcell suspension to the flasks as follows:

    ______________________________________                                                    MEDIA  CELLS                                                      ______________________________________                                        25 cm.sup.2   4.0 ml   0.5-1.0 ml                                             ______________________________________                                    

Incubate the cultures at 37° C. in a humid, 5% CO₂ enriched atmosphere.Leave the caps loose for CO₂ exchange.

DNA Analysis:

If requested by the physician, save at least 1 ml for DNA. (NOTE: Thecells do not need to be alive for DNA testing.) Label the tube with thepatient name, date, specimen type, and tracking number and place in theDNA beaker in the refrigerator.

Liquid Nitrogen:

Prepare a minimum of 2 cryovials for long-term storage in liquidnitrogen according to the freezing protocol.

Immunomodulation:

If requested by the physician, save at least 2.0 ml for the assay andstore refrigerated until the IM assay is performed.

Reporting Results:

Within 24 hours after setting up the pharmacosensitivity, read the cellcontrol and abrin plates under the inverted scope. Scan the plates inentirety. Count any clumps of cells and record as a background count onthe worksheet. Always scan the entire plate for colonies and aggregatesusing a hand tally counter to record the number of each. If no coloniesor aggregates are present, count the number of cells in four squares.Divide by 4 to obtain the average number of cells in one square,multiply by 15, then by 13.36 to obtain the number of cells in theentire 35 mm plate. Use scientific notation to report the colonies,aggregates and/or cells for any number greater than 3 digits.Representative colonies, aggregates, and/or cells must bephotomicrographed and logged in the log book prior to discarding plates.Verify that controls fall within expected range. If not, document in thecomment section and notify the Supervisor immediately. Do not discardthe assay; it may be accepted at the discretion of the Medical Director.

Experiment 1 Activity of N. sativa on bone marrow cells

Preparation of N. sativa:

1. Dried seeds are finely ground, and 5 grams (5 g) of the powder isextracted with 95% ethanol 3 times, by soxhlet extraction. A Wheatonsoxhlet extraction apparatus consists of 3 parts: the bottom part is theflask in which the 95% ethanol is heated; the middle portion is theextractor; and the top the Allihn condenser, in which there is an inletand outlet for running cold water through the condenser. Dried N. sativaseed powder is packed in filter paper and placed in the extractor.

2. The pooled extracts are evaporated under reduced pressure to a knownvolume of 10 ml and loaded onto a silica gel (Keiselgel 60: Fluka)column and eluted with 95% methanol/water (9:1). In the column, silicagel suspended in methanol/water is layered on sand. N. sativa extract isadded on top of the silica gel, followed by the eluting solvent.

3. The active fraction (as indicated by brown color) is collected andseparated by preparing thin layer chromatography (TLC). On commerciallyavailable chromatography plates, a band of N. sativa active compound isspotted and run in a TLC jar using chloroform as the solvent system.

4. The active spot at the solvent front is removed, eluted with methanol(overnight), and evaporated under reduced pressure to render the productalcohol free.

5. The extract is then available as a soluble solution and is thenstored at 2° C.

The approximate content of the active compound in the seed in 2.2%(w/w).

Calculation of active compound in N. sativa extract proceeded asfollows: the active compound present in seed extract is 2.2% w/w. Onegram of seeds was added to 10 ml assay media. Therefore, active compoundadded was 22 mg/ml media. Finally 40 μl from this was taken for assay.Hence, the active compound actually added was 88 μg. This was furtherdiluted 1:10, 1:100 and 1:1000 to have a working concentration of 8.8μg, 880 ng and 88 ng respectively.

Colony Forming Unit Assay

Specimen: Bone marrow sufficient to yield an optimum of 5.0 mlmononuclear cells at a concentration of 1.0×10⁶ cells/ml was collected.

Assay: The assay was performed according to a modified process Metcalf(1984, 1985), which is incorporated herein by reference. The process isdescribed in detail above in Procedure 2. To each 25 cm² flask, 1×10⁶bone marrow cells are added, and incubated at 37° C. with 5% CO₂ and 100μl of N. sativa plant extract.

Immunofluorescence Staining of Cells: The peripheral blood cells werecollected aseptically. For every panel of analysis 3.0 ml whole bloodwas needed. The assay was conducted according to processes disclosed inand partially modified from Karen (1989) and Merkel et al. (1987), whichare incorporated herein by reference. The process is described in detailabove in Procedure 1.

The N. sativa plant extract was incubated with peripheral blood cellsfrom cancer patients over an 18 hour period at 37° C. in a 5% CO₂incubator. Gates for the antibodies CD3, CD19, HLADR, LAK CD3+/CD56+, NKCD3-/CD56+, CD38, CD37, and CD33 surface markers were set on a BectonDickinson FacScan serial No. 81326. These antibodies are designed toassist in the characteristics of the types of cells. The clusterdesignation (CD) is used to group two or more mABs that havestatistically similar expression on normal and neoplastic cells and celllines, as well as similar recognition of the same antigen or bindingsite (epitope). While mABs in a CD group usually define differentepitopes on the same antigen, some CD groups, primarily non-lymphoidclusters, contain mABs that do not bind to the same antigen.

The results obtained from Experiment 1 were compared with that of bonemarrow growth factors and biological response modifiers namely GM-CSF,G-CSF, erythropoietin, interferon, IL-2 and STS.

At days 7, 14 and 21, each flask was scanned under a Reichart JungBiostar inverted microscope. A colony is defined as 40 or more cellsadhering to the bottom of the flask.

The colony forming cell unit (CFU) assay on 1×10⁶ bone marrow cellsobtained from 2 cancer patients was performed with and withoutincubation with 100 μl of N. sativa plant extract.

Results: The colony forming cell unit assay on 1×10⁶ bone marrow cellspost 15 days incubation with 100 μl of plant extract showed a 600%elevation. This elevation was 125% after 21 days of incubation. Amongthe different growth factors/biological response modifiers, IL-2 andGM-CSF gave the maximum elevation of 120 and 115% after incubation withbone marrow cells for 15 days. There was not much detection of CFUelevation post 21-day incubation with growth factor/biological responsemodifiers. Interferon showed marginal elevation after a 15-dayincubation, but negligible detectable response after 21 days.

N. sativa plant extract helps restore the immune competent cells inimmunosuppressed cancer patients. The colony forming cell unit assayindicates increase in CFU count when bone marrow cells from cancerpatients were incubated with N. sativa plant extract as well as withdifferent growth factors indicating that N. sativa plant extracts mimicthe potential application of these several growth factors. Even thoughthe plant extract was used without dissolving, it had no toxic effectsagainst human bone marrow cells. Hence, N. sativa plant extract helpsrestore the immune competent cells in immunosupressed cancer patientsand over-stimulates bone marrow in normal individuals.

Experiment 2 Correlation Between Elevation in Immune CompetentCells/Hematopoiesis and N. sativa Extract Immunomodulatory Testing

Immunomodulatory testing evaluates the activity of patients' sera andwhite blood cells in relation to interferon, interleukin and lymphokinedemonstrating the presence of interferon inhibitor factor and lymphokineinhibitor factor in the experimental procedure. A detailed descriptionof immunomodulatory testing is found above in Procedure 3.

To test for possible correlation between elevation in immune competentcells/hematopoiesis and N. sativa extract, the various surface markersof immune competent cells were analyzed by automated flow cytometry asdescribed above in Procedure 3.

The results indicate an elevation in CD19, HLADR, NK CD3-/CD56+ and CD38population. There was not much alteration in CD3, CD37 and CD33 surfacemarker index. A proliferation of connective tissue `L` cells wasobserved upon incubation with cancer patient serum and N. sativa extractat 37° centigrade in 5% CO₂ incubator for 7 days.

Cell Line: The mouse connective tissue L929 cells were obtained from theATCC and grown in MEM medium (GIBCO) with 20% fetal equine serum(BioWhittaker) as described above.

Assay: The assay was conducted according to the process described inMedenica, et al. (1990), which is incorporated herein by reference.Proliferation of 5×10⁵ `L` cells/ml was checked on a double layer agarprocedure in which lower 1.5% agar layer media contained 125 ml FBS, 125ml 2× G.G. fortified solution and 250 ml 1× G.G. fortified solution. Theupper layer stock media contained 150 ml FBS, 150 ml 2× G.G. fortifiedsolution and 150 ml 1× G.G. fortified solution. To 2.0 ml of upperlayer, 0.1 ml of N. sativa extract, 0.3 ml of `L` cells and 0.6 ml of1.5% agar was added to make a total volume 3.0 ml. Growth of `L` cellswere analyzed under an inverted microscope type Reichert Jung biostarpost incubation at 37° centigrade and 5% CO₂ after 7 days.

The results are illustrated in Table 4 as follows:

                  TABLE 4                                                         ______________________________________                                                           CELL      %                                                INCUBATION MIXTURE NUMBER    ELEVATION                                        ______________________________________                                        `L` CELLS & MEDIA  2.0 × 10.sup.4                                                                     0                                               `L` CELLS & IFN & MEDIA                                                                          2.7 × 10.sup.4                                                                    35                                               `L` CELLS & IL-2 & MEDIA                                                                         2.3 × 10.sup.4                                                                    15                                               `L` CELLS & SERUM & PLANT                                                                        4.7 × 10.sup.4                                                                    135                                              EXTRACT                                                                       `L` CELLS & WBC & PLANT                                                                          7.4 × 10.sup.4                                                                    270                                              EXTRACT                                                                       ______________________________________                                    

Connective tissue `L` cells at a concentration of 5×10⁵ were incubatedwith IFN, IL-2 or plant extract at 37° C. in a humid 5% CO₂ incubatorfor 7 days. The SD of the data was <10%.

Results: Peripheral blood cells from cancer patients upon incubationwith N. sativa extract over an 18-hour period at 37° C. in 5% CO₂incubator showed elevation in CD19, HLADR, NK CD3-/CD56+ and CD38populations as shown in FIG. 1. CD19 surface marker index showed anelevation of 90% as compared to control, while HLADR, NKCD3-/CD56+ andCD38 population showed elevations of 44, 38 and 5% respectively. Therewas not much alteration in the CD3 population.

N. sativa extract, rather than the increase in immune competent cellnumber, helped free tumor antigen binding sites on B cells, therebyelevating the CD19 and associated cell population. Data from flowcytometric analysis indicates elevation in CD19, HLADR, NK CD3-/CD56+and CD38 population. There was not much alteration in CD3, CD37 and CD33surface marker index. This data clearly indicates proliferation inimmune competent cells, mainly the humoral response against tumorantigens. Thus, the extract, rather than the increase in immunecompetent cell number, helps free tumor antigen binding sites on Bcells, thereby elevating the CD19 and associated cell population.

Connective tissue `L` cells at a concentration of 5×10⁵ upon incubationwith N. sativa plant extract and patient serum/white blood cells showedelevation in cell number. `L` cells when incubated with media alone was2.0×10⁴, which increased to 4.7×10⁴ and to 7.4×10⁴ upon incubation withcancer patient serum and plant extract and cancer patient white bloodcells and plant extract, respectively. This indicated an elevation of135 and 270% respectively as shown in Table 4 (supra.) On the otherhand, interferon and interleukin-2 gave marginal elevations of 35 and15% only upon incubation with 5×10⁵ `L` cells in an ideally sameenvironment as that of N. sativa plant extract.

Immunomodulatory evaluation indicates enhanced proliferation of theconnective tissue `L` cells in presence of N. sativa extract than in itsabsence, thereby indicating positive immunomodulatory effect of theextract. Similar results were also obtained with patient white bloodcells.

Data indicates presence of an interferon inhibitor factor (IIF) incancer patient serum due to which there is no proliferation of the `L`cells when they were incubated with patient serum alone. IIF andlymphokine inhibitor factor (LIF) in patient serum get suppressed posttreatment with N. sativa plant extract, thereby exhibiting enhancedproliferation of `L` cells. The IIF cannot be identified with antibodiesagainst interferon.

IIF is a factor which does not allow the production of autologous (self)interferon in the human body. When the inducer initiates the productionof interferon, the human body produces another protein which neutralizesthe possibility for production of interferon. LIF includes all factorsthat suppress cytokine activity, including interferons.

The increased stimulation of connective cells by the reduction of IIFand LIF allows the human body to produce its own interferon, which theninterferes with controlling connective tissue cell production. For thisreason, N. sativa has potential use in connective tissue diseases suchas rheumatoid arthritis, lupus and autoimmune diseases.

Experiment 3 Cytopathic Effects of Vesicular Stomatitis Virus

Cell Line: The human amnion or "WISH" cells were obtained from theAmerican Type Cell Culture facility (ATCC) and grown in Basal EagleMedium (BEM) (GIBCO) with 15% fetal bovine serum.

Virus: Vesicular stomatitis virus (VSV) was obtained from the ATCC. TheVSV stock solution was diluted 1000× with BEM containing 2% fetal bovineserum (FBS).

Assay: The assay was conducted as described above in Procedure 4. Fordetermining the amount of protection conferred to the "WISH" cells by N.sativa plant extract, complete viral cytopathic effect (CPE) wascalculated by inoculating 8-10 ml per flask of the 1:1000 VSV into eachWISH cell flask (containing 3.5×10⁵ cells/ml) such that it covered theentire cell layer. The flasks were incubated for 1 hour at 37° C. in a5% CO₂ incubator. Fifteen ml of the BMEM with 2% FBS were added to eachflask and incubated at 37° C. in a 5% CO₂ incubator for 1-3 days oruntil cell layer showed complete viral cytopathic effect (CPE). N.sativa plant extract was serially diluted in a 96-well polystyreneplate. To 40 μl of the extract was added 100 μl of the 3.5×10⁵ cells/ml"WISH" cell suspension and the plates were incubated for approximately18-24 hours at 37° C. at 5% CO₂. In the control wells, no extract wasadded. Next day (after 18-24 hours) the plates were observed under NikonTMS microscope for confluency. Fifty μl VSV were added (in aconcentration to yield 100% CPE), and Incubated 24-48 hours at 37° C.with 5% CO₂.

The next day, the plates were observed under a Nikon TMS microscope for100% CPE in the viral control wells. Once satisfied, the plates weredumped and blotted. One hundred μl of 15% neutral red dye were added toall wells. The wells were incubated at 37° C. in 5% CO₂ incubator for 45minutes. The plates were dumped, blotted and rinsed with 200 μl PBS atpH 6.85. The plates were again dumped and blotted. The cells were elutedby adding 100 μl of the eluting solution to each well. The plates wereread on Bio-Rad Model 3550 microplate reader.

The concentration of vesicular stomatitis virus (VSV) producing 100%cytopathic effect (CPE) of human amniotic "WISH" cells was firstdetermined, which was 1×10⁵ plaque forming units. Optimum protection toWISH cells conferred by N. sativa extract was then calculated byserially diluting N. sativa extract and adding in between WISH cells andVSV.

Results: The results depicted in FIG. 2 indicate protection of "WISH"cells in the order of 25 and 65% upon incubation with 1:10 and 1:100diluted N. sativa plant extract. There was no noticeable cytopathiceffect when N. sativa was added at a dilution of 1:1000. Approximateactive compound concentrations were calculated according to materialsand methods. Serum interferon levels were measured after plant extractadministration according a modified procedure of Finter (1969).

Protection of Human Amniotic "WISH" cells from cytopathic effects ofvesicular stomatitis virus (VSV) was observed upon administration of1:1000 diluted N. sativa plant extract. This had an active compoundconcentration of 88 ng. At a lower dilution, there was some effect.Theoretically, a patient weighing 70 kg has about 7×10¹³ cells in itsbody and N. sativa will be useful in the dosage of 20-40 g to protectagainst viral attack in virus endemic areas. Upon administration of theextract, the serum interferon level is found to increase and hence N.sativa has interferon-like antiviral activity. The extract possiblyblocks all receptor sites on "WISH" cells which are essential forattachment of the virus to cell membrane.

Experiment 4 Pharmacosensitivity Assay

The tumor specimens can be of 2 types: solid tumor and liquid tumor.

Solid tumor: 2 grams of tumor specimen were excised, cut into pea sizeportions and gently homogenized in a Potter-Elvehjam homogenizer with10% FBS, centrifuged at 1600 rpm for 20 minutes in a Mistral 3000irefrigerated centrifuge at 4° C. Cells were finally suspended in mediaRPMI 1640 with 10% FBS into a fine suspension.

Liquid tumor: The specimen was collected from the ascitic fluid ofcancer patients. The ascitic fluid was gently layered on a histopaqueusing a 1:1 ratio of the histopaque to specimen, and centrifuged at 1600rpm for 20 minutes in a Mistral 3000i refrigerated centrifuge at 4° C.Cells were finally suspended in RPMI with 10% FBS.

Assay: The assay was performed according to the modified processes ofVon Hoff, et al. (1981) and Salmon, et al. (1978), which areincorporated herein by reference, as described above in Procedure 5.Tumoricidal activity of N. sativa was checked on a double agar layer.The lower layer of warm 2.5% Bacto-agar working solution was poured into35 mm grid petri dishes. The plates were then allowed to sit undisturbedon a flat surface until the agar solidified. To 2.1 ml of the upperlayer media containing 150 ml FBS, 150 ml 2× G.G. fortified solution and150 ml 1× G.G. fortified solution, 0.3 ml of the tumor cells and 0.6 mlof the 1.5% agar were added.

The upper layer was carefully layered on top of the lower layer in the35 mm grid petri dish. Tumor specimens grown without the extract andwith `Abrin` were used as controls. Tumor cell growth in the presence ofthe plant extract was monitored 7, 14 and 21 days post incubation at 37°C. and 5% CO₂, at which time the cells were counted under aReichert-Jung biostar inverted microscope. Cells from tumor specimens[8.2×10⁴ ] were grown with media/Abrin plant extract on a double agarlayer and incubated for 7, 14 and 21 days at 37° C. in a humid 5% CO₂incubator. The results are shown on Table 5 as follows:

                  TABLE 5                                                         ______________________________________                                        INCU-                                                                         BATION INCUBATION TIME                                                        MIX-            %              %            %                                 TURE   7 days   Inh    14 days Inh  21 days Inh                               ______________________________________                                        TUMOR  8.2 × 10.sup.4                                                                   0      10.0 × 10.sup.4                                                                 0    10.5 × 10.sup.4                                                                 0                                 CELL &                                                                        MEDIA                                                                         TUMOR  8.1 × 10.sup.4                                                                   1.2    9.8 × 10.sup.4                                                                  2.0  10.3 × 10.sup.4                                                                 1.9                               CELL &                                                                        ABRIN                                                                         TUMOR  5.3 × 10.sup.4                                                                   35.0   5.4 × 10.sup.4                                                                  46.0  5.5 × 10.sup.4                                                                 48.0                              CELL &                                                                        EX-                                                                           TRACT                                                                         ______________________________________                                    

The SD of the data was <10%. Percentage of inhibition was calculated asdescribed in Materials & Methods.

The results are analyzed on the basis of pharmacosensitivity assay inwhich tumor cell growth was monitored. Seven, 14 and 21 days postincubation with plant extract indicated tumoricidal activity. Hence, N.sativa extract possessed a multifaceted toxic-free cure for cancer.

Results: Tumor specimens (2 grams) either from solid tumor masses ormalignant ascites were mechanically homogenized into a suspensioncontaining 8.2×10⁴ cells. Incubation of tumor cells at 37° C. in a humid5% CO₂ incubator for 7, 14 and 21 days with N. sativa plant extractindicated a percent inhibition of 35, 46 and 48, respectively.Incubation with Abrin in an ideally similar condition did not exhibitany noticeable inhibition.

N. sativa promotes anti-tumor activity. Data from pharmacosensitivityscreening indicates anti-tumor activity of N. sativa plant extract, ascell growth in culture was inhibited anywhere between 25-90%. In vitrotumor study report, using rapid screen for early pharmacosensitivityscreening, in which cells were incubated with N. sativa plant extractfor 2 hours, gave similar results. Hence, the extract indicatesanti-tumor function mainly against melanoma and colon cancer types. N.sativa plant extract destroys tumor cells and leaves normal cells alone,possibly because of its ability to bind to cell surface asialofeutin(lectin) in diseased cells, which causes aggregation and clumping oftumor cells. It also blocks enzymes and inappropriate gene productsinvolved in nucleic acid synthesis and metabolism.

It is understood that the invention is not confined to the particularconstruction and arrangement herein described, but embraces suchmodified forms thereof as come within the scope of the claims followingthe bibliographic citations.

BIBLIOGRAPHY

Agarwal, R., Kharya, M. D., and Shrivastava, R., 1979, "Antimicrobialanthelmintic activities of the essential oil of Nigella sativa Linn,"Indian J. Exp. Biol. 17, 1264.

Akhtar, M. S.; Riffat, S., 1991, "Field Trial of Saussurea Lappa RootsAgainst Nematodes and Nigella Sativa Seeds Against Cestodes inChildren," JPMA J Pak Med Assoc 41(8)185-7.

al-Awadi, F. M., Khattar, M. A. and Gumaa, K. A., 1985, "On theMechanism of the Hypoglycemic Effect of a Plant Extract," Diabetologia28, 432-434.

al-Awadi, F.; Fatania, H.; Shamte, U., 1991, "The Effect of a Plant'sMixture Extract on Liver Gluconeogenesis in Streptozotocin InducedDiabetic Rats," Diabetes Res (Scotland) 18(4):163-8.

Aruna, K.; Sivaramakrishnan, V. M., 1990, "Plant Products as ProtectiveAgents Against Cancer," Indian J Exp Biol 28(11):1008-11.

Aruna, K.; Sivaramakrishnan, V. M., 1992, "Anticarcinogenic Effects ofSome Indian Plant Products," Food Chem Toxicol (England) 30(11)953-6.

Bitterman, W. A.; Farhadian, H.; Abu Samra, C.; Lerner, D.; Amoun, H.;Krapf, D;. Makov, U. E., 1991, "Environmental and Nutritional FactorsSignificantly Associated with Cancer of the Urinary Tract amongDifferent Ethnic Groups," Urol Clin North Am 18(3)501-8.

Chopra, R. N., Chopra, I. C., Handa, K. L., and Kapur, L. D., 1982,Indigenous Drugs of India, Academic Publishers; Calcutta, India.

Datta, A. K., Biswas, A. K. and Ghosh, P. D., 1983, "Chromosomalvariations in callus tissues of two species of Nigella sativa, L.," Thenucleus 26(3) 173-177.

Elkadi, A ; Kandil, O., 1987, "The Black Seed Nigella sativa andImmunity Its Effects on Human Cell Subsets," Fed Proc 45(4)1222.

Finter, N. B., 1969, "Dye Uptake Methods for Assessing ViralCytopathogenicity and Their Application to Interferon Assays, " J. Gen.Virol. 5, 419-427.

Karen, D. F., 1989, "Flow Cytometry in Clinical Diagnosis," AmericanSociety of Clinical Pathology (press).

Kirtikar, K. R. and Basu, B. D., 1982, Indian Medicinal Plants, Vol. I,Bishen Singh and Mahendra Pal Singh, eds., Dehra Dun, India.

Kumar, B. H. and Thakur, S. S., 1989, "Effect of Certain Non-edible SeedOils on Growth Regulation in Disdercus Similis (F), J. Anim. Morphol.Physiol. 36(2) pp. 209-218.

Medenica, R., Alonso, K., Huschart, T. and Tyler, K., 1990, "TumorTissue Culture for Determining Efficient Drug for Intra-Arterial,Intra-Hepatic Chemotherapy and Interferon of Colon Carcinoma LiverMetastasis," Abstract presented at Conference on Combining BRM withCytotoxic in the Treatment of Cancer.

Merkel, D. E., Dressier, L. G. and McGuire, W. L., 1987, "Flow CytometryCellular DNA Contents and Prognosis in Human Malignancy," J. of ClinicalOncology, 5, 1690-1703.

Metcalf, D., 1984, Clonal Culture of Hematopoietic Cells, Elsevier/NorthAmerican Biomedical Press.

Metcalf, D., 1985, "The Granulocyte Macrophage Colony StimulatingFactors," Science 229, 16-22.

Nadkarni, K. M., 1976, "Crocus sativus, Nigella sativa," Indian MateriaMedica, K. M. Nadkarni (ed) Bombay, India; Popular Prakashan, Vol 1, pg.386-411.

Nair, S. C.; Salomi, M. J.; Panikkar, B.; Pannikar, K. R., 1991,"Modulatory Effects of Crocus sativus and Nigella sativa Extracts onCisplatin-Induced Toxicity in Mice," J Ethnopharmocol 31(1): 75-83.

Salmon, S. E., Hamburger, A. W., Soehnlein, B., 1978, "Quantitation ofDifferential Sensitivity of Human Tumor Stem Cells to Anticancer Drugs,, " N. Eng. J. Med. 298, 1321-1327.

Salomi, N. J.; Nair, S. C.; Jayawardhanan, K. K.; Varghese, C. D.;Panikkar, K. R., 1992, "Anititumour Principles from Nigella sativaSeeds," Cancer Lett 63(1):41-6.

Salomi, M. J.; Nair, S. C.; Panikkar, K. R., 1991, "Inhibitory Effectsof Nigella sativa and Saffron (Crocus Sativus) on ChemicalCarcinogenesis in Mice," Nutr Cancer 16(1):67-72.

Sayed, M. D., "Traditional Medicine in Health Care," 1980, JEthnopharmocol 2(1):19-22.

Shayeb, N. A. and Mabrouk, S. S., 1984, "Utilization of Some Edible andMedicinal Plants to Inhibit Aflatoxin Formation," Nutrition ReportsInternational 29(2).

Siddiqui, M. B., Alam, M. M., Husain, W. and Sharma, G. K., 1988,"Ethno-medical Study of Plants used for Terminating Pregnancy,"Fitoterapia V. LIX, no. 3, 250.

Salmon, S. E.; Hamburger, A. W.; Soehnlein, B.; et al., 1978,"Quantitation of differential sensitivity of human tumor stem cells toanticancer drugs," N Engl J Med 298:1321-1327.

Srivastava, K. C., 1989, "Extracts from Two Frequently ConsumedSpices--Cumin (Cuminum cyminum) and Turmeric (Curcuma Longa)--InhibitPlatelet Aggregation and Alter Eicosanoid Biosynthesis in Human BloodPlatelets," Prostaglandins Leukot Essent Fatty Acids 37(1)57-64.

Tennekoon, K. H.; Jeevathayaparan, S.; Kurukulasooriya, A. P.;Karunayake, E. H., 1991, "Possible Hepatotoxicity of Nigella sativaSeeds and Dregea Volubilis Leaves," J Ethnopharmocol 31(3):283-9.

Vihan, V. S. and Panwar, H. S., 1987, "Galactopoietic Effect of Nigellasativa (H-Kalonji) in Clinical Cases of Agalactia in Goats, " IndianVet. J. 64, 347-349.

Von Hoff, D. D., Cowan, J., Harris, J. and Reisdorf, G., 1981, "HumanTumor Cloning: Feasibility and Clinical Correlations," Cancer Chemother.Pharmacol. 6, 265-271.

I claim:
 1. A method for activating immune competent cells in humans inorder to increase the immune function in humans, the immune competentcells being selected from the group consisting of CD19, HLADR,NKCD3-/CD56+ and CD38, the method comprising administering to humans aneffective dose of an extract from Nigella sativa at a concentrationwhich is effective to activate the immune competent cells by reducingthe presence of interferon inhibitor factor or lymphokine inhibitorfactor.
 2. The method of claim 1 wherein the effective dose is betweenabout 20 and about 40 grams of the extract per day.
 3. The method ofclaim 1 wherein the effective dose is about 30 grams of the extract perday.
 4. A method for increasing antibody producing B cells in humans,the B cells having minor antigen binding sites, comprising administeringto humans an effective dose of an extract from Nigella sativa, at aconcentration which is effective to free the tumor antigen binding siteson the B cells thereby increasing the antibody producing B cells.
 5. Themethod of claim 4 wherein the effective dose is between about 20 andabout 40 grams of extract per day.
 6. The method of claim 4 wherein theeffective dose is about 30 grams of extract per day.